Nucleic acid molecules encoding a transmembrane serine protease 10, the encoded polypeptides and methods based thereon

ABSTRACT

Provided herein are type II transmembrane serine protease 10 (MTSP10) polypeptides. Zymogen and activated forms of these polypeptides as well as single and two chain forms of the protease domain are also provided. Methods using the polypeptides to identify compounds that modulate the activity of an MTSP10 are provided.

RELATED APPLICATIONS

Benefit of priority under 35 U.S.C. § 119(e) is claimed to U.S.provisional application Ser. No. 60/291,001, filed May 14, 2001, EdwinL. Madison and Yeh, Jiunn-Chern, entitled “NUCLEIC ACID MOLECULESENCODING TRANSMEMBRANE SERINE PROTEASE 10, THE ENCODED PROTEINS ANDMETHODS BASED THEREON.” The subject matter of this application isincorporated in its entirety by reference thereto.

FIELD OF INVENTION

Nucleic acid molecules that encode proteases and portions thereof,particularly protease domains are provided. Also provided areprognostic, diagnostic and therapeutic methods using the proteases anddomains thereof and the encoding nucleic acid molecules.

BACKGROUND OF THE INVENTION AND OBJECTS THEREOF

Cancer, which is a leading cause of death in the United States, ischaracterized by an increase in the number of abnormal neoplastic cells,which proliferate to form a tumor mass, the invasion of adjacent tissuesby these neoplastic tumor cells, and the generation of malignant cellsthat metastasize via the blood or lymphatic system to regional lymphnodes and to distant sites. Among the hallmarks of cancer is a breakdownin the communication among tumor cells and their environment. Normalcells do not divide in the absence of stimulatory signals and ceasedividing in the presence of inhibitory signals. Growth-stimulatory andgrowth-inhibitory signals, are routinely exchanged between cells withina tissue. In a cancerous, or neoplastic, state, a cell acquires theability to “override” these signals and to proliferate under conditionsin which normal cells do not grow.

In order to proliferate tumor cells acquire a number of distinctaberrant traits reflecting genetic alterations. The genomes of certainwell-studied tumors carry several different independently altered genes,including activated oncogenes and inactivated tumor suppressor genes.Each of these genetic changes appears to be responsible for impartingsome of the traits that, in the aggregate, represent the full neoplasticphenotype.

A variety of biochemical factors have been associated with differentphases of metastasis. Cell surface receptors for collagen, glycoproteinssuch as laminin, and proteoglycans, facilitate tumor cell attachment, animportant step in invasion and metastases. Attachment triggers therelease of degradative enzymes which facilitate the penetration of tumorcells through tissue barriers. Once the tumor cells have entered thetarget tissue, specific growth factors are required for furtherproliferation. Tumor invasion and progression involve a complex seriesof events, in which tumor cells detach from the primary tumor, breakdown the normal tissue surrounding it, and migrate into a blood orlymphatic vessel to be carried to a distant site. The breaking down ofnormal tissue barriers is accomplished by the elaboration of specificenzymes that degrade the proteins of the extracellular matrix that makeup basement membranes and stromal components of tissues.

A class of extracellular matrix degrading enzymes has been implicated intumor invasion. Among these are the matrix metalloproteinases (MMP). Forexample, the production of the matrix metalloproteinase stromelysin isassociated with malignant tumors with metastatic potential (see, e.g.,McDonnell et al. (1990) Smnrs. in Cancer Biology 1:107–115; McDonnell etal. (1990) Cancer and Metastasis Reviews 9:309–319).

The capacity of cancer cells to metastasize and invade tissue isfacilitated by degradation of the basement membrane. Several proteinaseenzymes, including the MMPs, have been reported to facilitate theprocess of invasion of tumor cells. MMPs are reported to enhancedegradation of the basement membrane, which thereby permits tumorouscells to invade tissues. For example, two major metalloproteinaseshaving molecular weights of about 70 kDa and 92 kDa appear to enhanceability of tumor cells to metastasize.

Type II Transmembrane Serine Proteases

In addition to the MMPs, serine proteases have been implicated inneoplastic disease progression. Most serine proteases, which are eithersecreted enzymes or are sequestered in cytoplasmic storage organelles,have roles in blood coagulation, wound healing, digestion, immuneresponses and tumor invasion and metastasis. A class of cell surfaceproteins designated type II transmembrane serine proteases, which aremembrane-anchored proteins with additional extracellular domains, hasbeen identified. As cell surface proteins, they are positioned to play arole in intracellular signal transduction and in mediating cell surfaceproteolytic events.

Cell surface proteolysis is a mechanism for the generation ofbiologically active proteins that mediate a variety of cellularfunctions. Membrane-associated proteases include membrane-typemetallo-proteinases (MT-MMP), ADAMs (proteases that containdisintegrin-like and metalloproteinase domains) and the transmembraneserine proteases. In mammals, at least 17 members of the transmembraneserine protease family are known, including seven in humans (see, Hooperet al. (2001) J. Biol. Chem. 276:857–860). These include: corin(accession nos. AF133845 and AB013874; see, Yan et al. (1999) J. Biol.Chem. 274:14926–14938; Tomia et al. (1998) J. Biochem. 124:784–789; Uanet al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97:8525–8529);enterpeptidase (also designated enterokinase; accession no. U09860 forthe human protein; see, Kitamoto et al. (1995) Biochem. 27:4562–4568;Yahagi et al. (1996) Biochem. Biophys. Res. Commun. 219:806–812;Kitamoto et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91:7588–7592;Matsushima et al. (1994) J. Biol. Chem. 269:19976–19982;); human airwaytrypsin-like protease (HAT; accession no. AB002134; see Yamaoka et al.J. Biol. Chem. 273:11894–11901); MTSP1 and matriptase (also calledTADG-15; see SEQ ID Nos. 1 and 2; accession nos. AF133086/AF118224,AF04280022; Takeuchi et al. (1999) Proc. Natl. Acad. Sci. U.S.A.96:11054–1161; Lin et al. (1999) J. Biol. Chem. 274:18231–18236;Takeuchi et al. (2000) J. Biol. Chem. 275:26333–26342; and Kim et al.(1999) Immunogenetics 49:420–429); hepsin (see, accession nos. M18930,AF030065, X70900; Leytus et al. (1988) Biochem. 27: 11895–11901; Vu etal. (1997) J. Biol. Chem. 272:31315–31320; and Farley et al. (1993)Biochem. Biophys. Acta 1173:350–352; and see, U.S. Pat. No. 5,972,616);TMPRS2 (see, Accession Nos. U75329 and AF113596; Paoloni-Giacobino etal. (1997) Genomics 44:309–320; and Jacquinet et al. (2000) FEBS Lett.468:93–100); and TMPRSS4 (see, Accession No. NM 016425; Wallrapp et al.(2000) Cancer 60:2602–2606).

Serine proteases, including transmembrane serine proteases and secretedproteases, have been implicated in processes involved in neoplasticdevelopment and progression. While the precise, detailed mechanism bywhich these proteases promote tumor growth and progression has not beenelaborated, serine proteases and inhibitors thereof are involved in thecontrol of many intra- and extracellular physiological processes,including degradative actions in cancer cell invasion, metastaticspread, and neovascularization of tumors, that are involved in tumorprogression. It is believed that proteases are involved in thedegradation of extracellular matrix (ECM) and contribute to tissueremodeling, and are necessary for cancer invasion and metastasis. Theactivity and/or expression of some proteases have been shown tocorrelate with tumor progression and development.

For example, a membrane-type serine protease MTSP1 (also calledmatriptase; see SEQ ID Nos. 1 and 2 from U.S. Pat. No. 5,972,616; andGenBank Accession No. AF118224; (1999) J. Biol. Chem. 274:18231–18236;U.S. Pat. No. 5,792,616; see, also Takeuchi (1999) Proc. Natl. Acad.Sci. U.S.A. 96:11054–1161) that is expressed in epithelial cancer andnormal tissue (Takeucuhi et al. (1999) Proc. Natl. Acad. Sci. USA96:11054–61) has been identified. Matriptase was originally identifiedin human breast cancer cells as a major gelatinase (see, U.S. Pat. No.5,482,848) and was initially believed to be a type of matrixmetalloprotease (MMP). It has been proposed that it plays a role in themetastasis of breast cancer. Matriptase also is expressed in a varietyof epithelial tissues with high levels of activity and/or expression inthe human gastrointestinal tract and the prostate. MTSPs, designatedMTSP3, MTSP4, MTSP6 have been decribed in published International PCTapplication No. WO 01/57194, based in International PCT application No.PCT/US01/03471.

Prostate-specific antigen (PSA), a kallikrein-like serine protease,degrades extracellular matrix glycoproteins fibronectin and laminin,and, has been postulated to facilitate invasion by prostate cancer cells(Webber et al. (1995) Clin. Cancer Res., 1(10):1089–94). Blocking PSAproteolytic activity with PSA-specific monoclonal antibodies results ina dose-dependent decrease in vitro in the invasion of the reconstitutedbasement membrane Matrigel by LNCaP human prostate carcinoma cells whichsecrete high levels of PSA.

Hepsin, a cell surface serine protease identified in hepatoma cells, isoverexpressed in ovarian cancer (Tanimoto et al. (1997) Cancer Res.,57(14):2884–7). The hepsin transcript appears to be abundant incarcinoma tissue and is almost never expressed in normal adult tissue,including normal ovary. It has been suggested that hepsin is frequentlyoverexpressed in ovarian tumors and therefore can be a candidateprotease in the invasive process and growth capacity of ovarian tumorcells.

A serine protease-like gene, designated normal epithelial cell-specific1 (NES1) (Liu et al., Cancer Res., 56(14):3371–9 (1996)) has beenidentified. Although expression of the NES1 mRNA is observed in allnormal and immortalized nontumorigenic epithelial cell lines, themajority of human breast cancer cell lines show a drastic reduction or acomplete lack of its expression. The structural similarity of NES1 topolypeptides known to regulate growth factor activity and a negativecorrelation of NES1 expression with breast oncogenesis suggest a director indirect role for this protease-like gene product in the suppressionof tumorigenesis.

Hence transmembrane serine proteases appear to be involved in theetiology and pathogenesis of tumors. There is a need to furtherelucidate their role in these processes and to identify additionaltransmembrane proteases. Therefore, it is an object herein to providetransmembrane serine protease (MTSP) proteins and nucleic acids encodingsuch MTSP proteases that are involved in the regulation of orparticipate in tumorigenesis and/or carcinogenesis. It is also an objectherein to provide prognostic, diagnostic and therapeutic screeningmethods using such proteases and the nucleic acids encoding suchproteases.

SUMMARY

Provided herein is the protease domain of a protein designated herein asMTSP10. The protease domain and full-length protein, including thezymogen and activated forms, and uses thereof are also provided.Proteins encoded by splice variants are also provided. Hence, providedherein is a family proteins designated MTSP10, and functional domains,including one or more of a transmembrane (TM) domain, two CUB domains,three LDL receptor type a domains and a serine protease catalyticdomain, especially protease (or catalytic) domains thereof. Alsoproivded are muteins and other derivatives and analogs thereof. Alsoprovided herein are nucleic acids encoding the MTSP10s.

The protease domains provided herein include, but are not limited to,the single chain region having an N-terminus at the cleavage site foractivation of the zymogen, through the C-terminus, or C-terminaltruncated portions thereof that exhibit proteolytic activity as asingle-chain polypeptide in vitro proteolysis assays of MTSP10, from amammal, including a human, that, for example, displays functionalactivity in tumor cells that is different from its activity non-tumorcells.

Nucleic acid molecules encoding the proteins and protease domains arealso provided. Nucleic acid molecules that encode a single-chainprotease domain or catalytically active portion thereof and also thosethat encode the full-length MTSP10 or portions thereof are provided. Inone embodiment, a nucleic acid that encodes a MTSP, designated MTSP10 isprovided. The nucleic acid molecule includes the sequence of nucleotidesset forth in SEQ ID No. 5 or SEQ ID No. 22 or a portion thereof (see,also EXAMPLE 1) that encodes a catalytically active polypeptide or adomain thereof.

Also provided are nucleic acid molecules that encode all or a portionencoding a catalytically active polypeptide, or a nucleic acid moleculethat encodes the protease domain or a larger polypeptide that caninclude up to the full length polypeptide and that hybridizes to suchMTSP10-encoding nucleic acid along their full-length or along at leastabout 70%, 80% or 90% of the full-length and encode the protease domainor portion thereof are provided. Hybridization is generally effectedunder conditions of at least low, generally at least moderate, and oftenhigh stringency.

The isolated nucleic acid fragment is DNA, including genomic or cDNA, oris RNA, or can include other components, such as protein nucleic acid orother nucleotide analogs. The isolated nucleic acid may includeadditional components, such as heterologous or native promoters, andother transcriptional and translational regulatory sequences, thesegenes may be linked to other genes, such as reporter genes or otherindicator genes or genes that encode indicators.

Also provided is an isolated nucleic acid molecule that includes thesequence of molecules that is complementary to the nucleotide sequenceencoding MTSP10 or the portion thereof.

Also provided are nucleic acid molecules that hybridize under conditionsof at least low stringency, generally moderate stringency, moretypically high stringency to the sequence of nucleotides set forth inSEQ ID No. 5 or SEQ ID No. 22 or degenerates thereof. In one embodiment,the isolated nucleic acid fragment hybridizes to a nucleic acid moleculecontaining the nucleotide sequence set forth in SEQ ID No. 5 or SEQ IDNo. 22 (or degenerates thereof) under high stringency conditions. In oneembodiment, it contains the sequence of nucleotides set forth in SEQ IDNo. 5. A full-length MTSP10 polypeptide includes the sequence of aminoacids set forth in SEQ ID No. 6 or SEQ ID No. 23, and is encoded by asequence of nucleotides set forth in SEQ ID No. 5 or SEQ ID No. 22 ordegenerates thereof. Methods for isolating nucleic acid encoding otherMTSP10s, including nucleic acid molecules encoding full-length moleculesand splice variants and MTSPs from species, such as cows, sheep, goats,pigs, horses, primates, including chimpanzees and gorillas, rodents,dogs, cats and other species of interest, such as domesticated animals,farm and zoo animals are also provided. The nucleic acid moleculesprovided herein, including those set forth in SEQ ID Nos. 5 and 23 canbe used to obtain nucleic acid molecules encoding full-length MTSP10polypeptides from human sources or from other species, such as byscreening appropriate libraries using the nucleic acid molecules orselected primers or probes based thereon.

Also provided are fragments thereof or oligonucleotides that can be usedas probes or primers and that contain at least about 10, 14, 16nucleotides, generally less than 1000 or less than or equal to 100, setforth in SEQ ID No. 5 or SEQ ID No. 22 (or the complement thereof); orcontain at least about 30 nucleotides (or the complement thereof) orcontain oligonucleotides that hybridize along their full-length (or atleast about 70, 80 or 90% thereof) to any such fragments oroligonucleotides. The length of the fragments are a function of thepurpose for which they are used and/or the complexity of the genome ofinterest. Generally probes and primers contain less than about 30, 50,150 or 500 nucleotides.

Also provided are plasmids containing any of the nucleic acid moleculesprovided herein. Cells containing the plasmids are also provided. Suchcells include, but are not limited to, bacterial cells, yeast cells,fungal cells, plant cells, insect cells and animal cells.

Methods of expressing the encoded MTSP10 polypeptide and portionsthereof using the cells are also provided, as are cells that expressMTSP10 on the cell surface. Such cells are used in methods ofidentifying candidate therapeutic compounds.

MTSP10, particularly the protease domain thereof, can be produced bygrowing the above-described cells under conditions whereby the MTSP10 isexpressed by the cells, and recovering the expressed MTSP10 polyeptide.

Also provided are cells, generally eukaryotic cells, such as mammaliancells and yeast cells, in which the MTSP10 polypeptide is expressed onthe surface of the cells. Such cells are used in drug screening assaysto identify compounds that modulate the activity of the MTSP10polypeptide. These assays, including in vitro binding assays, andtranscription based assays in which signal transduction mediateddirectly or indirectly, such as via activation of pro-growth factors, bythe MTSP10 is assessed.

Also provided are peptides that are encoded by such nucleic acidmolecules. Included among those polypeptides are the MTSP10 proteasedomain or a polypeptide with amino acid changes such that thespecificity and/or protease activity remains substantially unchanged. Inparticular, a substantially purified mammalian MTSP10 polypeptide isprovided that includes a serine protease catalytic domain and mayadditionally include other domains. The MTSP10 can form homodimers andcan also form heterodimers with some other protein, such as amembrane-bound protein. Also provided is a substantially purifiedprotein including a sequence of amino acids that has at least 60%, 70%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% identity to the MTSP10 where the percentageidentity is determined using standard algorithms and gap penalties thatmaximize the percentage identity. A human MTSP10 polypeptide isexemplified, although other mammalian MTSP10 polypeptides arecontemplated. Splice variants of the MTSP10, particularly those with aproteolytically active protease domain, are contemplated herein.

In other embodiments, substantially purified polypeptides that include aprotease domain of a MTSP10 polypeptide or a catalytically activeportion thereof are provided. Among these are polypeptides that includea sequence of amino acids that has at least 60%, 70%, 80%, 85%, 90%, 95%or 100% sequence identity to SEQ ID No. 6 or SEQ ID No. 23 or to aportion thereof that includes a catalytically active polyeptide or a CUBdomain or an LDL receptor domain.

Also provided are muteins of the single chain protease domain of MTSP10particularly muteins in which the Cys residue in the protease domainthat is free (i.e., does not form disulfide linkages with any other Cysresidue in the protease domain) is substituted with another amino acidsubstitution, typically, although not necessarily, with a conservativeamino acid substitution or a substitution that does not eliminate theactivity, and muteins in which a glycosylation site(s) is eliminated.Hence muteins in which one or more of the Cys residues, particularly, aresidue that is paired in the activated two form, but unpaired in theprotease domain alone (ie., the Cys a residue position 26 (see SEQ IDNos. 5 and 6) in the protease domain), is/are replaced with any aminoacid, typically, although not necessarily, a conservative amino acidresidue, such as Ser, are contemplated. Muteins of MTSP10, particularlythose in which Cys residues, such as the unpaired Cys in the singlechain protease domain, is replaced with another amino acid that does noteliminate the activity, are provided. Muteins in which otherconservative or non-conservative amino acid substitutions in whichcatalytic activity is retained are also contemplated (see, e.g., Table1, for exemplary amino acid substitutions).

MTSP10 polypeptides, including, but not limited to splice variantsthereof, and nucleic acids encoding MTSPs, and domains, derivatives andanalogs thereof are provided herein. Single chain protease domains thathave an N-terminus functionally equivalent to that generated byactivation of the zymogen form of MTSP10 are also provided. The cleavagesite for the protease domain of MTSP10 is between amino acid R and aminoacids I (R↓IIGGT) (see SEQ ID NO. 6, residues 1–5; residues 462–467 SEQID No. 23) There are potential glycosylation sites at N₆₇, N₂₇₂; N₃₃₆;N₃₈₃, N₄₀₉ and N₄₁₈ SEQ ID No. 23). The catalytic triad of the MTSP10 inSEQ ID No. 23 is H₅₀₃, D₅₅₁ and S₆₄₇.

There are potential disulfide bonds as follows: C₄₈₈–C₅₀₄, C₅₈₇–C₆₅₃;C₆₁₉–C₆₃₂; C₆₄₃–C₆₇₃ (see SEQ ID Nos. 22 and 23) (chymotrypsin numbering42 to 58; 136–201; 168–182 and 191–220). Disulfide bonds form betweenthe Cys residues C₅₇₃–C₂₉₆ to link the protease domain to another domainso that upon activation cleavage (between residues R₄₆₂ and I₄₆₃ of SEQID No. 23) the resulting polypeptide is a two chain molecule. The C₅₇₃(SEQ ID NO. 23, is a free Cys in the single chain form of the proteasedomain. As noted the protease also can be provided as a two chainmolecule. Single chain and two chain forms are proteolytically active. Atwo chain form of the polypeptide set forth in SEQ ID No. 23 isprovided; smaller catalytically active two chain forms are alsoprovided. A two chain form is produced by bonding, typically between theC₅₇₃ and a Cys outside the protease domain, such as Cys₂₉₆. Uponactiation cleavage the bond remains resulting in a two chainpolypeptide. The size of chain “A” is a function the starting length ofthe polypeptide prior to activation cleavage between the R₄₆₂ and I₄₆₃.Any length polyeptide that includes the protease domain (residues463–692 of SEQ ID No. 23) or catalytically active fragments thereof, iscontemplated herein. Two chain forms include at least the proteasedomain a polypeptide from C₂₉₆ up to and including C₅₇₃.

MTSPs are expressed or are activated in certain tumor or cancer cellssuch as lung, prostate, colon and breast, ovarian, pancreatic, lung inother tumors. MTSP10 is of interest because it is expressed or is activein tumor cells. In particular, it is shown herein, that MTSP10 is, forexample, expressed in esophageal tumor tissues, in lung carcinoma,prostate cancers, pancreatic and breast cancers and in cell lines aswell as in certain normal cells and tissues (see e.g., EXAMPLES fortissue-specific expression profile). The level of activated MTSP10 canbe diagnostic of prostate, uterine, lung esophagus, or colon cancer orleukemia or other cancer. The expression and/or activation of MTSP10 onor in the vicinity of a cell or in a bodily fluid in a subject can be amarker for breast, prostate, lung, colon and other cancers.

Hence the MTSPs provided herein can serve as diagnostic markers forcertain tumors. In certain embodiments, the MTSP10 polypeptide isdetectable in a body fluid at a level that differs from its level inbody fluids in a subject not having a tumor. In other embodiments, thepolypeptide is present in a tumor; and a substrate or cofactor for thepolypeptide is expressed at levels that differ from its level ofexpression in a non-tumor cell in the same type of tissue. In otherembodiments, the level of expression and/or activity of the MTSP10polypeptide in tumor cells differs from its level of expression and/oractivity in non-tumor cells. In other embodiments, the MTSP10 is presentin a tumor; and a substrate or cofactor for the MTSP10 is expressed atlevels that differ from its level of expression in a non-tumor cell inthe same type of tissue.

Assays for identifying effectors, such as compounds, including smallmolecules, and conditions, such pH, temperature and ionic strength, thatmodulate the activation, expression or activity of MTSP10 are alsoprovided herein. In exemplary assays, the effects of test compounds onthe ability of a protease domain of MTSP10 to proteolytically cleave aknown substrate, typically a fluorescently, chromogenically or otherwisedetectably labeled substrate, are assessed. Agents, generally compounds,particularly small molecules, that modulate the activity of the proteasedomain are candidate compounds for modulating the activity of theMTSP10. The protease domains can also be used to produceprotease-specific antibodies.

Also provided are methods for screening for compounds that modulate theactivity of MTSP10. The compounds are identified by contacting them withthe MTSP10 or protease domain thereof and a substrate for the MTSP10. Achange in the amount of substrate cleaved in the presence of thecompounds compared to that in the absence of the compound indicates thatthe compound modulates the activity of the MTSP10. Such compounds areselected for further analyses or for use to modulate the activity of theMTSP10, such as inhibitors or agonists. The compounds can also beidentified by contacting the substrates with a cell that expresses theMTSP10 or the extracellular domain or proteolytically active portionthereof.

Also provided herein are methods of modulating the activity of theMTSP10 and screening for compounds that modulate, including inhibit,antagonize, agonize or otherwise alter the activity of the MTSP10. Ofparticular interest is the extracellular domain of MTSP10 that includesthe proteolytic (catalytic) portion of the protein.

Additionally provided herein are antibodies that specifically bind tosingle and two chains forms of MTSP10, cells, combinations, kits andarticles of manufacture that contain the antibodies. Antibodies thatspecifically bind to the MTSP10, particularly the single-chain proteasedomain, the two-chain form of the protease domain, the zymogen andactivated form of MTSP10 and other fragments thereof. Neutralizingantibodies that inhibit a biological activity, particularly proteaseactivity are also provided.

Further provided herein are prognostic, diagnostic, therapeuticscreening methods using MTSP10 and the nucleic acids encoding MTSP10. Inparticular, the prognostic, diagnostic and therapeutic screening methodsare used for preventing, treating, or for finding agents useful inpreventing or treating, tumors or cancers such as lung carcinoma, colonadenocarcinoma and ovarian carcinoma.

Also provided herein are modulators of the activity of MTSP10,especially the modulators obtained according to the screening methodsprovide herein. Such modulators can have use in treating cancerousconditions.

Methods of diagnosing a disease or disorder characterized by detectingan aberrant level of an MTSP10 in a subject is provided. The method canbe practiced by measuring the level of the DNA, RNA, protein orfunctional activity of the MTSP10. An increase or decrease in the levelof the DNA, RNA, protein or functional activity of the MTSP, relative tothe level of the DNA, RNA, protein or functional activity found in ananalogous sample not having the disease or disorder (or other suitablecontrol) is indicative of the presence of the disease or disorder in thesubject or other relative any other suitable control.

Also provided are methods of identifying a compound that binds to thesingle-chain and/or two-chain form of MTSP10, by contacting a testcompound with a both forms; determining to which form the compoundbinds; and if it binds to a form of MTSP10, further determining whetherthe compound has at least one of the following properties:

-   -   (i) inhibits activation of the single-chain zymogen form of        MTSP10;    -   (ii) inhibits activity of the two-chain or single-chain form;        and    -   (iii) inhibits dimerization of the protein.        The forms can be full length or truncated forms, including but        not limited to, the protease domain resulting from cleavage at        the activation cleavage site (between amino acids R₄₆₂ and I₄₆₃        SEQ ID No. 23); or from expression of the protease domain or        catalytically active portions thereof.

Pharmaceutical composition containing the protease domain and/orfull-length or other domain of an MTSP10 polypeptide are provided hereinin a pharmaceutically acceptable carrier or excipient are providedherein.

Also provided are articles of manufacture that contain MTSP10polypeptide and protease domains of MTSP10 in single chain forms oractivated forms. The articles contain a) packaging material; b) thepolypeptide (or encoding nucleic acid), particularly the single chainprotease domain thereof; and c) a label indicating that the article isfor using ins assays for identifying modulators of the activities of anMTSP10 polypeptide is provided herein.

Conjugates containing a) an MTSP10 polypeptide or protease domain in asingle or two chain form; and b) a targeting agent linked to the MTSPdirectly or via a linker, wherein the agent facilitates: i) affinityisolation or purification of the conjugate; ii) attachment of theconjugate to a surface; iii) detection of the conjugate; or iv) targeteddelivery to a selected tissue or cell, is provided herein. The conjugatecan contain a plurality of agents linked thereto. The conjugate can be achemical conjugate; and it can be a fusion protein. The targeting agentcan be a protein or peptide fragment. The protein or peptide fragmentcan include a protein binding sequence, a nucleic acid binding sequence,a lipid binding sequence, a polysaccharide binding sequence, or a metalbinding sequence.

Combinations, kits and articles of manufacture containing the MTSP10polypeptides, domains thereof, or encoding nucleic acids are alsoprovided herein. For example, combinations are provided herein. Thecombination can include: a) an inhibitor of the activity of an MTSP10;and b) an anti-cancer treatment or agent. The MTSP inhibitor and theanti-cancer agent can be formulated in a single pharmaceuticalcomposition or each is formulated in a separate pharmaceuticalcomposition. The MTSP10 inhibitor can be an antibody or a fragment orbinding portion thereof made against the MTSP10, such as an antibodythat specifically binds to the protease domain, an inhibitor of MTSP10production, or an inhibitor of MTSP10 membrane-localization or aninhibitor of MTSP10 activation. Other MTSP10 inhibitors include, but arenot limited to, an antisense nucleic acid or double-stranded RNA(dsRNA), such as RNAi, encoding the MTSP10, particularly a portion ofthe protease domain; a nucleic acid encoding at least a portion of agene encoding the MTSP10 with a heterologous nucleotide sequenceinserted therein such that the heterologous sequence inactivates thebiological activity encoded MTSP10 or the gene encoding it. For example,the portion of the gene encoding the MTSP10 can flank the heterologoussequence to promote homologous recombination with a genomic geneencoding the MTSP10.

Also provided are methods for treating or preventing a tumor or cancerin a mammal by administering to a mammal an effective amount of aninhibitor of an MTSP10, whereby the tumor or cancer is treated orprevented. The MTSP10 inhibitor used in the treatment or for prophylaxisis administered with a pharmaceutically acceptable carrier or excipient.The mammal treated can be a human. The treatment or prevention methodcan additionally include administering an anti-cancer treatment or agentsimultaneously with or subsequently or before administration of theMTSP10 inhibitor.

Also provided are transgenic non-human animals bearing inactivated genesencoding the MTSP and bearing the genes encoding the MTSP10 undernon-native promotor control are provided. Such animals are useful inanimal models of tumor initiation, growth and/or progression models.Transgenic non-human animals containing heterolgous nucleic acid MTSP10under native, non-native promotor control or on an exogenous element,such as a plasmid or artificial chromosome, are additionally providedherein. In particular, recombinant non-human animals are providedherein, where the gene of an MTSP10 is under control of a promoter thatis not the native promoter of the gene or that is not the nativepromoter of the gene in the non-human animal or where the nucleic acidencoding the MTSP10 is heterologous to the non-human animal and thepromoter is the native or a non-native promoter or the MTSP10 is on anextrachromosomal element, such as a plasmid or artificial chromosome.Recombinant and transgenic animals can be produced by homologousrecombination and non-homologous recombination methods.

Methods of gene therapy are provided. Such methods can be effectedadministering in vivo or ex vivo an inactivating form of the MTSP10 orby administering an MTSP-encoding nucleic acid molecule are alsoprovided.

Also provided are methods of treatments of tumors by administering aprodrug that is activated by MTSP10 that is expressed or active in tumorcells, particularly those in which its functional activity in tumorcells is greater than in non-tumor cells. The prodrug is administeredand, upon administration, active MTSP10 expressed on cells cleaves theprodrug and releases active drug in the vicinity of the tumor cells. Theactive anti-cancer drug accumulates in the vicinity of the tumor. Thisis particularly useful in instances in which MTSP10 is expressed oractive in greater quantity, higher level or predominantly in tumor cellscompared to other cells.

Also provided are methods of diagnosing the presence of a pre-malignantlesion, a malignancy, or other pathologic condition in a subject, byobtaining a biological sample from the subject; exposing it to adetectable agent that binds to a two-chain and/or single-chain form ofMTSP10, where the pathological condition is characterized by thepresence or absence of the two-chain and/or single-chain form.

Methods of inhibiting tumor invasion or metastasis or treating amalignant or pre-malignant condition by administering an agent thatinhibits activation of the zymogen form of MTSP10 or an activity of theactivated form are provided. The conditions include, but are not limitedto, a condition, such as a tumor, of the breast, cervix, prostate, lung,ovary or colon.

DETAILED DESCRIPTION

A. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the invention(s) belong. All patents, patent applications,published applications and publications, Genbank sequences, websites andother published materials referred to throughout the entire disclosureherein, unless noted otherwise, are incorporated by reference in theirentirety. In the event that there are a plurality of definitions forterms herein, those in this section prevail. Where reference is made toa URL or other such indentifier or address, it understood that suchidentifiers can change and particular information on the internet cancome and go, but equivalent information can be found by searching theinternet. Reference thereto evidences the availability and publicdissemination of such information.

As used herein, the abbreviations for any protective groups, amino acidsand other compounds, are, unless indicated otherwise, in accord withtheir common usage, recognized abbreviations, or the IUPAC-IUBCommission on Biochemical Nomenclature (see, (1972) Biochem.11:942–944).

As used herein, serine protease refers to a diverse family of proteaseswherein a serine residue is involved in the hydrolysis of proteins orpeptides. The serine residue can be part of the catalytic triadmechanism, which includes a serine, a histidine and an aspartic acid inthe catalysis, or be part of the hydroxyl/∈-amine or hydroxyl/α-aminecatalytic dyad mechanism, which involves a serine and a lysine in thecatalysis. Of particular interest are SPs of mammalian, including human,origin. Those of skill in this art recognize that, in general, singleamino acid substitutions in non-essential regions of a polypeptide donot substantially alter biological activity (see, e.g., Watson et al.(1987) Molecular Biology of the Gene, 4th Edition, The Bejacmin/CummingsPub. co., p. 224).

As used herein, “transmembrane serine protease (MTSP)” refers to afamily of transmembrane serine proteases that share common structuralfeatures as described herein (see, also Hooper et al. (2001) J. Biol.Chem. 276:857–860). Thus, reference, for example, to “MTSP” encompassesall proteins encoded by the MTSP gene family, including but are notlimited to: MTSP3, MTSP4, MTSP6, MTSP7 or an equivalent moleculeobtained from any other source or that has been prepared syntheticallyor that exhibits the same activity. Other MTSPs include, but are notlimited to, corin, enterpeptidase, human airway trypsin-like protease(HAT), MTSP1, TMPRSS2 and TMPRSS4. Sequences of encoding nucleic acidmolecules and the encoded amino acid sequences of exemplary MTSPs and/ordomains thereof are set forth, for example in U.S. application Ser. No.09/776,191 (SEQ ID Nos. 1–12, 49, 50 and 61–72 therein, published asInternational PCT application No. WO 01/57194). The term also encompassMTSPs with amino acid substitutions that do not substantially alteractivity of each member and also encompasses splice variants thereof.Suitable substitutions, including, although not necessarily,conservative substitutions of amino acids, are known to those of skillin this art and can be made without eliminating the biological activity,such as the catalytic activity, of the resulting molecule.

As used herein an MTSP10, whenever referenced herein, includes at leastone or all of or any combination of:

-   -   a polypeptide encoded by the sequence of nucleotides set forth        in SEQ ID No. 5 or SEQ ID No. 22 or by a sequence of nucleotides        that includes nucleotides that encode the sequence of amino        acids set forth in SEQ ID No. 6 or SEQ ID No. 23;    -   a polypeptide encoded by a sequence of nucleotides that        hybridizes under conditions of low, moderate or high stringency        to the sequence of nucleotides set forth in is set forth as SEQ        ID No. 5 or SEQ ID No. 22;    -   a polypeptide that includes the sequence of amino acids set        forth in SEQ ID No. 6 or SEQ ID No. 23 or a catalytically active        portion thereof;    -   a polypeptide that includes a sequence of amino acids having at        least about 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,        87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or        99% sequence identity with the sequence of amino acids set forth        in SEQ ID No. 6 or SEQ ID No. 23; and/or    -   a polypeptide encoded by a splice variant of the MTSP10 that        includes the sequence of amino acids set forth in SEQ ID No. 6        or SEQ ID No. 23.

In particular, MTSP10 polypeptides, with a protease domain as indicatedin SEQ ID Nos. 5 and 6, is provided. The polypeptide is a single or twochain polypeptide. Smaller portions thereof that retain proteaseactivity are also provided. The protease domains from MTSPs vary in sizeand constitution, including insertions and deletions in surface loops.They retain conserved structure, including at least one of the activesite triad, primary specificity pocket, oxyanion hole and/or otherfeatures of serine protease domains of proteases. Thus, for purposesherein, the protease domain is a portion of an MTSP, as defined herein,and is homologous to a domain of other MTSPs, such as corin,enterpeptidase, human airway trypsin-like protease (HAT), MTSP1,TMPRSS2, and TMPRSS4, which have been previously identified; it was notrecognized, however, that an isolated single chain form of the proteasedomain could function proteolytically in in vitro assays. As with thelarger class of enzymes of the chymotrypsin (S1) fold (see, e.g.,Internet accessible MEROPS data base), the MTSPs protease domains sharea high degree of amino acid sequence identity. The His, Asp and Serresidues necessary for activity are present in conserved motifs. Theactivation site, which results in the N-terminus of the second chain inthe two chain form is located in a conserved motif and readily can beidentified. In the exemplified MTSP10, it is between residues R₄₆₂ andI₄₆₃ of SEQ ID No. 23.

The MTSP10 can be from any animal, particularly a mammal, and includesbut are not limited to, primates, including humans, rodents, fowl,ruminants and other animals. The full-length zymogen or two-chainactivated form is contemplated or any domain thereof, including theprotease domain, which can be a two-chain activated form, or a singlechain form.

As used herein, a “protease domain of an MTSP” refers to anextracellular protease domain of an MTSP that exhibits proteolyticactivity and shares homology and structural features with thechymotrypsin/trypsin family protease domains. Hence it is at least theminimal portion of the domain that exhibits proteolytic activity asassessed by standard in vitro assays. Contemplated herein are suchprotease domains and catalytically active portions thereof. Alsoprovided are truncated forms of the protease domain that include thesmallest fragment thereof that acts catalytically as a single chainform.

A protease domain of an MTSP10, whenever referenced herein, includes atleast one or all of or any combination of or a catalytically activeportion of:

-   -   a) a polypeptide that includes the sequence of amino acids set        forth in SEQ ID No. 6, particularly amino acids 1–230 thereof;    -   b) a polypeptide that includes the sequence of amino acids set        forth in SEQ ID No. 23;    -   c) a polypeptide encoded by a sequence of nucleotides that        hybridizes under conditions of low, moderate or high stringency        to the sequence of nucleotides set forth in SEQ ID No. 5 or to        SEQ ID No. 22;    -   d) a polypeptide that includes the sequence of amino acids set        forth in SEQ ID No. 6 or SEQ ID No. 23 or a catalytically active        portion thereof;    -   e) a polypeptide that includes a sequence of amino acids having        at least about 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,        87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or        99% sequence identity with the sequence of amino acids set forth        in SEQ ID No. 6 or SEQ ID No. 23; and/or    -   f) a protease domain of a polypeptide encoded by a splice        variant of a sequence of nucleotides that encodes an MTSP10 of        any of a)–e).

The protease domains of MTSPs vary in size and constitution, includinginsertions and deletions in surface loops. They retain conservedstructure, including at least one of the active site triad, primaryspecificity pocket, oxyanion hole and/or other features of serineprotease domains of proteases. Thus, for purposes herein, the proteasedomain is a portion of an MTSP, as defined herein, and is homologous toa domain of other MTSP. As with the larger class of enzymes of thechymotrypsin (S1) fold (see, e.g., Internet accessible MEROPS database), the MTSP protease domains share a high degree of amino acidsequence identity. The His, Asp and Ser residues necessary for activityare present in conserved motifs. The activation site, whose cleavagecreates the N-terminus of the protease domain in the two-chain forms islocated in a conserved motif and readily can be identified.

By active form is meant a form active in vivo and/or in vitro. Asdescribed herein, the protease domain also can exist as a two-chainform. It is shown herein that, at least in vitro, the single chain formsof the SPs and the catalytic domains or proteolytically active portionsthereof (typically C-terminal truncations) exhibit protease activity.Hence provided herein are isolated single chain forms of the proteasedomains of SPs and their use in in vitro drug screening assays foridentification of agents that modulate the activity thereof.

As used herein, the catalytically active domain of an MTSP refers to theprotease domain. Reference to the protease domain of an MTSP generallyrefers to the single chain form of the protein. If the two-chain form orboth forms is intended, it is so-specified. The zymogen form of eachprotein is a single chain, which is converted to the active two chainform by activation cleavage.

As used herein, activation cleavage refers to the cleavage of theprotease at the N-terminus of the protease domain (generally between anR and I in the full-length protein, which includes amino acid residues1–5 of SEQ ID No. 6, residue 463–467 of SEQ ID No. 23). By virtue of theCys-Cys pairing between a Cys outside the protease domain and a Cys inthe protease domain (in this instance Cys₅₇₃ SEQ ID No. 23, uponcleavage the resulting polypeptide has two chains (“A” chain and the “B”chain, which is the protease domain). Cleavage can be effected byanother protease or autocatalytically.

As used herein, a two-chain form of the protease domain refers to atwo-chain form that is formed from the two-chain form of the protease inwhich the Cys pairing between, in this instance, a Cys outside theprotease domain and Cys₅₇₃ (SEQ ID No. 23), which links the proteasedomain to the remainder of the polypeptide, the “A” chain. A two chainprotease domain form refers to any form in which the “remainder of thepolypeptide”, i.e., “A” chain, is shortened and includes from at the Cysoutside the protease domain. For example a two chain form of an MTSP10includes from Cys₂₉₆ up to and including Cys₅₇₃ of SEQ ID No. 23 wherethe A chain includes Cys₂₉₆ to R₄₆₂ and the B chain includes I₄₆₃ to atleast Cys₅₇₃.

MTSPs of interest include those that are activated and/or expressed intumor cells different, typically higher, from those in non-tumor cells;and those from cells in which substrates therefor differ in tumor cellsfrom non-tumor cells or differ with respect to the substrates,co-factors or receptors, or otherwise alter the activity or specificityof the MTSP.

As used herein, a human protein is one encoded by nucleic acid, such asDNA, present in the genome of a human, including all allelic variantsand conservative variations as long as they are not variants found inother mammals.

As used herein, a “nucleic acid encoding a protease domain orcatalytically active portion of a SP” shall be construed as referring toa nucleic acid encoding only the recited single chain protease domain oractive portion thereof, and not the other contiguous portions of the SPas a continuous sequence.

As used herein, catalytic activity refers to the activity of the SP as aserine protease. Function of the SP refers to its function in tumorbiology, including promotion of or involvement in initiation, growth orprogression of tumors, and also roles in signal transduction. Catalyticactivity refers to the activity of the SP as a protease as assessed inin vitro proteolytic assays that detect proteolysis of a selectedsubstrate.

As used herein, a CUB domain is a motif that mediates protein-proteininteractions in complement components C1r/C1s and has also beenidentified in various proteins involved in developmental processes.

As used herein, (LDLR) refers to a low density lipoprotein receptordomain, which mediate binding to an LDL receptor.

As used herein, a zymogen is an inactive precursor of a proteolyticenzyme. Such precursors are generally larger, although not necessarilylarger than the active form. With reference to serine proteases,zymogens are converted to active enzymes by specific cleavage, includingcatalytic and autocatalytic cleavage, or by binding of an activatingco-factor, which generates an active enzyme. A zymogen, thus, is anenzymatically inactive protein that is converted to a proteolytic enzymeby the action of an activator.

As used herein, “disease or disorder” refers to a pathological conditionin an organism resulting from, e.g., infection or genetic defect, andcharacterized by identifiable symptoms.

As used herein, neoplasm (neoplasia) refers to abnormal new growth, andthus means the same as tumor, which can be benign or malignant. Unlikehyperplasia, neoplastic proliferation persists even in the absence ofthe original stimulus.

As used herein, neoplastic disease refers to any disorder involvingcancer, including tumor development, growth, metastasis and progression.

As used herein, cancer refers to a general term for diseases caused byany type of malignant tumor.

As used herein, malignant, as applies to tumors, refers to primarytumors that have the capacity of metastasis with loss of growth controland positional control.

As used herein, an anti-cancer agent (used interchangeable with“anti-tumor or anti-neoplastic agent”) refers to any agents used in theanti-cancer treatment. These include any agents, when used alone or incombination with other compounds, that can alleviate, reduce,ameliorate, prevent, or place or maintain in a state of remission ofclinical symptoms or diagnostic markers associated with neoplasticdisease, tumor and cancer, and can be used in methods, combinations andcompositions provided herein. Non-limiting examples of anti-neoplasticagents include anti-angiogenic agents, alkylating agents,antimetabolites, certain natural products, platinum coordinationcomplexes, anthracenediones, substituted ureas, methylhydrazinederivatives, adrenocortical suppressants, certain hormones, antagonistsand anti-cancer polysaccharides.

As used herein, a splice variant refers to a variant produced bydifferential processing of a primary transcript of genomic nucleic acid,such as DNA, that results in more than one type of mRNA. Splice variantsof SPs are provided herein.

As used herein, angiogenesis is intended to broadly encompass thetotality of processes directly or indirectly involved in theestablishment and maintenance of new vasculature (neovascularization),including, but not limited to, neovascularization associated withtumors.

As used herein, anti-angiogenic treatment or agent refers to anytherapeutic regimen and compound, when used alone or in combination withother treatment or compounds, that can alleviate, reduce, ameliorate,prevent, or place or maintain in a state of remission of clinicalsymptoms or diagnostic markers associated with undesired and/oruncontrolled angiogenesis. Thus, for purposes herein an anti-angiogenicagent refers to an agent that inhibits the establishment or maintenanceof vasculature. Such agents include, but are not limited to, anti-tumoragents, and agents for treatments of other disorders associated withundesirable angiogenesis, such as diabetic retinopathies, restenosis,hyperproliferative disorders and others.

As used herein, non-anti-angiogenic anti-tumor agents refer toanti-tumor agents that do not act primarily by inhibiting angiogenesis.

As used herein, pro-angiogenic agents are agents that promote theestablishment or maintenance of the vasculature. Such agents includeagents for treating cardiovascular disorders, including heart attacksand strokes.

As used herein, undesired and/or uncontrolled angiogenesis refers topathological angiogenesis wherein the influence of angiogenesisstimulators outweighs the influence of angiogenesis inhibitors. As usedherein, deficient angiogenesis refers to pathological angiogenesisassociated with disorders where there is a defect in normal angiogenesisresulting in aberrant angiogenesis or an absence or substantialreduction in angiogenesis.

As used herein, the protease domain of an SP protein refers to theprotease domain of an SP that exhibits proteolytic activity. Hence it isat least the minimal portion of the protein that exhibits proteolyticactivity as assessed by standard assays in vitro. It refers, herein, toa single chain form and also the two chain activated form (where the twochain form is intended it will be so-noted). Exemplary protease domainsinclude at least a sufficient portion of sequences of amino acids setforth in SEQ ID No. 6 (amino acis 1–230, encoded by nucleotides in SEQID No. 5) to exhibit protease activity.

Also contemplated are nucleic acid molecules that encode a polypeptidethat has proteolytic activity in an in vitro proteolysis assay and thathave at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity with the full-length or a protease domain of an MTSP10polypeptide or other domain thereof, or that hybridize along theirfull-length or along at least about 70%, 80% or 90% of the full-lengthto a nucleic acids that encode a protease domain or other domain,particularly under conditions of moderate, generally high, stringency.

For the protease domains, residues at the N-terminus can be critical foractivity. It is shown herein that the protease domain of the singlechain form of the MTSP10 protease is catalytically active. Hence theprotease domain generally requires the N-terminal amino acids thereoffor activity; the C-terminus portion can be truncated. The amount thatcan be removed can be determined empirically by testing the polypeptidefor protease activity in an in vitro assay that assesses catalyticcleavage.

Hence smaller portions of the protease domains, particularly the singlechain domains, thereof that retain protease activity are contemplated.Such smaller versions generally are C-terminal truncated versions of theprotease domains. Such domains exhibit conserved structure, including atleast one structural feature, such as the active site triad, primaryspecificity pocket, oxyanion hole and/or other features of serineprotease domains of proteases. Thus, for purposes herein, the proteasedomain is a single chain portion of an MTSP10, as defined herein, but ishomologous in its structural features and retention of sequence ofsimilarity or homology the protease domain of chymotrypsin or trypsin.The polypeptide exhibits proteolytic activity as a single chain.

As used herein, by homologous means about greater than 25% nucleic acidsequence identity, such as 25% 40%, 60%, 70%, 80%, 90% or 95%. Ifnecessary the percentage homology will be specified. The terms“homology” and “identity” are often used interchangeably. In general,sequences are aligned so that the highest order match is obtained (see,e.g.: Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, NewYork, 1991; Carillo et al. (1988) SIAM J. Applied Math 48:1073). Bysequence identity, the number of conserved amino acids are determined bystandard alignment algorithms programs, and are used with default gappenalties established by each supplier. Substantially homologous nucleicacid molecules would hybridize typically at moderate stringency or athigh stringency all along the length of the nucleic acid or or along atleast about 70%, 80% or 90% of the full-length nucleic acid molecule ofinterest. Also contemplated are nucleic acid molecules that containdegenerate codons in place of codons in the hybridizing nucleic acidmolecule.

Whether any two nucleic acid molecules have nucleotide sequences thatare at least, for example, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%“identical” can be determined using known computer algorithms such asthe “FAST A” program, using for example, the default parameters as inPearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programsinclude the GCG program package (Devereux, J., et al., Nucleic AcidsResearch 12(I):387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F., etal., J Molec Biol 215:403 (1990); Guide to Huge Computers, Martin J.Bishop, ed., Academic Press, San Diego, 1994, and Carillo et al. (1988)SIAM J Applied Math 48:1073). For example, the BLAST function of theNational Center for Biotechnology Information database can be used todetermine identity. Other commercially or publicly available programsinclude, DNAStar “MegAlign” program (Madison, Wis.) and the Universityof Wisconsin Genetics Computer Group (UWG) “Gap” program (MadisonWis.)). Percent homology or identity of proteins and/or nucleic acidmolecules can be determined, for example, by comparing sequenceinformation using a GAP computer program (e.g., Needleman et al. (1970)J. Mol. Biol. 48:443, as revised by Smith and Waterman ((1981) Adv.Appl. Math. 2:482). Briefly, the GAP program defines similarity as thenumber of aligned symbols (i.e., nucleotides or amino acids) which aresimilar, divided by the total number of symbols in the shorter of thetwo sequences. Default parameters for the GAP program can include: (1) aunary comparison matrix (containing a value of 1 for identities and 0for non-identities) and the weighted comparison matrix of Gribskov etal. (1986) Nucl. Acids Res. 14:6745, as described by Schwartz andDayhoff, eds., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, NationalBiomedical Research Foundation, pp. 353–358 (1979); (2) a penalty of 3.0for each gap and an additional 0.10 penalty for each symbol in each gap;and (3) no penalty for end gaps. Therefore, as used herein, the term“identity” represents a comparison between a test and a referencepolypeptide or polynucleotide.

As used herein, the term at least “90% identical to” refers to percentidentities from 90 to 99.99 relative to the reference polypeptides.Identity at a level of 90% or more is indicative of the fact that,assuming for exemplification purposes a test and referencepolynucleotide length of 100 amino acids are compared. No more than 10%(i.e., 10 out of 100) amino acids in the test polypeptide differs fromthat of the reference polypeptides. Similar comparisons can be madebetween a test and reference polynucleotides. Such differences can berepresented as point mutations randomly distributed over the entirelength of an amino acid sequence or they can be clustered in one or morelocations of varying length up to the maximum allowable, e.g. 10/100amino acid difference (approximately 90% identity). Differences aredefined as nucleic acid or amino acid substitutions, or deletions. Atthe level of homologies or identities above about 85–90%, the resultshould be independent of the program and gap parameters set; such highlevels of identity can be assessed readily, often without relying onsoftware.

As used herein, primer refers to an oligonucleotide containing two ormore deoxyribonucleotides or ribonucleotides, typically more than three,from which synthesis of a primer extension product can be initiated.Experimental conditions conducive to synthesis include the presence ofnucleoside triphosphates and an agent for polymerization and extension,such as DNA polymerase, and a suitable buffer, temperature and pH.

As used herein, animals include any animal, such as, but are not limitedto, goats, cows, deer, sheep, rodents, pigs and humans. Non-humananimals, exclude humans as the contemplated animal. The SPs providedherein are from any source, animal, plant, prokaryotic and fungal. MostMTSP10s are of animal origin, including mammalian origin.

As used herein, genetic therapy involves the transfer of heterologousnucleic acid, such as DNA, into certain cells, target cells, of amammal, particularly a human, with a disorder or conditions for whichsuch therapy is sought. The nucleic acid, such as DNA, is introducedinto the selected target cells in a manner such that the heterologousnucleic acid, such as DNA, is expressed and a therapeutic productencoded thereby is produced. Alternatively, the heterologous nucleicacid, such as DNA, can in some manner mediate expression of DNA thatencodes the therapeutic product, or it can encode a product, such as apeptide or RNA that in some manner mediates, directly or indirectly,expression of a therapeutic product. Genetic therapy can also be used todeliver nucleic acid encoding a gene product that replaces a defectivegene or supplements a gene product produced by the mammal or the cell inwhich it is introduced. The introduced nucleic acid can encode atherapeutic compound, such as a growth factor inhibitor thereof, or atumor necrosis factor or inhibitor thereof, such as a receptor therefor,that is not normally produced in the mammalian host or that is notproduced in therapeutically effective amounts or at a therapeuticallyuseful time. The heterologous nucleic acid, such as DNA, encoding thetherapeutic product can be modified prior to introduction into the cellsof the afflicted host in order to enhance or otherwise alter the productor expression thereof. Genetic therapy can also involve delivery of aninhibitor or repressor or other modulator of gene expression.

As used herein, heterologous nucleic acid is nucleic acid that (if DNAencodes RNA) and proteins that are not normally produced in vivo by thecell in which it is expressed or that mediates or encodes mediators thatalter expression of endogenous nucleic acid, such as DNA, by affectingtranscription, translation, or other regulatable biochemical processes.Heterologous nucleic acid, such as DNA, can also be referred to asforeign nucleic acid, such as DNA. Any nucleic acid, such as DNA, thatone of skill in the art would recognize or consider as heterologous orforeign to the cell in which is expressed is herein encompassed byheterologous nucleic acid; heterologous nucleic acid includesexogenously added nucleic acid that is also expressed endogenously.Examples of heterologous nucleic acid include, but are not limited to,nucleic acid that encodes traceable marker proteins, such as a proteinthat confers drug resistance, nucleic acid that encodes therapeuticallyeffective substances, such as anti-cancer agents, enzymes and hormones,and nucleic acid, such as DNA, that encodes other types of proteins,such as antibodies. Antibodies that are encoded by heterologous nucleicacid can be secreted or expressed on the surface of the cell in whichthe heterologous nucleic acid has been introduced. Heterologous nucleicacid is generally not endogenous to the cell into which it isintroduced, but has been obtained from another cell or preparedsynthetically. Generally, although not necessarily, such nucleic acidencodes RNA and proteins that are not normally produced by the cell inwhich it is now expressed.

As used herein, a therapeutically effective product for gene therapy isa product that is encoded by heterologous nucleic acid, typically DNA,that, upon introduction of the nucleic acid into a host, a product isexpressed that ameliorates or eliminates the symptoms, manifestations ofan inherited or acquired disease or that cures the disease. Alsoincluded are biologically active nucleic acid molecules, such as RNAiand antisense.

As used herein, recitation that a polypeptide consists essentially ofthe protease domain means that the only SP portion of the polypeptide isa protease domain or a catalytically active portion thereof. Thepolypeptide can optionally, and generally will, include additionalnon-SP-derived sequences of amino acids.

As used herein, cancer or tumor treatment or agent refers to anytherapeutic regimen and/or compound that, when used alone or incombination with other treatments or compounds, can alleviate, reduce,ameliorate, prevent, or place or maintain in a state of remission ofclinical symptoms or diagnostic markers associated with deficientangiogenesis.

As used herein, domain refers to a portion of a molecule, e.g., proteinsor the encoding nucleic acids, that is structurally and/or functionallydistinct from other portions of the molecule.

As used herein, protease refers to an enzyme catalyzing hydrolysis ofproteins or peptides. It includes the zymogen form and activated formsthereof. For clarity reference to protease refers to all forms, andparticular forms will be specifically designated. For purposes herein,the protease domain includes single and two chain forms of the proteasedomain of an SP protein. For MTSP10 the protease domain also includessingle and two chain forms of the protease domain.

As used herein, nucleic acids include DNA, RNA and analogs thereof,including peptide nucleic acids (PNA) and mixtures thereof. Nucleicacids can be single or double-stranded. When referring to probes orprimers, optionally labeled, with a detectable label, such as afluorescent or radiolabel, single-stranded molecules are contemplated.Such molecules are typically of a length such that their target isstatistically unique or of low copy number (typically less than 5,generally less than 3) for probing or priming a library. Generally aprobe or primer contains at least 14, 16 or 30 contiguous of sequencecomplementary to or identical a gene of interest. Probes and primers canbe 10, 20, 30, 50, 100 or more nucleic acids long.

As used herein, nucleic acid encoding a fragment or portion of an SPrefers to a nucleic acid encoding only the recited fragment or portionof SP, and not the other contiguous portions of the SP.

As used herein, operative linkage of heterologous nucleic acids toregulatory and effector sequences of nucleotides, such as promoters,enhancers, transcriptional and translational stop sites, and othersignal sequences refers to the relationship between such nucleic acid,such as DNA, and such sequences of nucleotides. Thus, operatively linkedor operationally associated refers to the functional relationship ofnucleic acid, such as DNA, with regulatory and effector sequences ofnucleotides, such as promoters, enhancers, transcriptional andtranslational stop sites, and other signal sequences. For example,operative linkage of DNA to a promoter refers to the physical andfunctional relationship between the DNA and the promoter such that thetranscription of such DNA is initiated from the promoter by an RNApolymerase that specifically recognizes, binds to and transcribes theDNA. In order to optimize expression and/or in vitro transcription, itcan be necessary to remove, add or alter 5′ untranslated portions of theclones to eliminate extra, potential inappropriate alternativetranslation initiation (i.e., start) codons or other sequences that caninterfere with or reduce expression, either at the level oftranscription or translation. Alternatively, consensus ribosome bindingsites (see, e.g., Kozak J. Biol. Chem. 266:19867–19870 (1991)) can beinserted immediately 5′ of the start codon and can enhance expression.The desirability of (or need for) such modification can be empiricallydetermined.

As used herein, a sequence complementary to at least a portion of anRNA, with reference to antisense oligonucleotides, means a sequencehaving sufficient complementarily to be able to hybridize with the RNA,generally under moderate or high stringency conditions, forming a stableduplex; in the case of double-stranded SP antisense nucleic acids, asingle strand of the duplex DNA (or dsRNA) can thus be tested, ortriplex formation can be assayed. The ability to hybridize depends onthe degree of complementarily and the length of the antisense nucleicacid. Generally, the longer the hybridizing nucleic acid, the more basemismatches with a SP encoding RNA it can contain and still form a stableduplex (or triplex, as the case can be). One skilled in the art canascertain a tolerable degree of mismatch by use of standard proceduresto determine the melting point of the hybridized complex.

For purposes herein, amino acid substitutions can be made in any of SPsand protease domains thereof provided that the resulting proteinexhibits protease activity. Amino acid substitutions contemplatedinclude conservative substitutions, such as those set forth in Table 1,which do not eliminate proteolytic activity. As described herein,substitutions that alter properties of the proteins, such as removal ofcleavage sites and other such sites are also contemplated; suchsubstitutions are generally non-conservative, but can be readilyeffected by those of skill in the art.

Suitable conservative substitutions of amino acids are known to those ofskill in this art and can be made generally without altering thebiological activity, for example enzymatic activity, of the resultingmolecule. Those of skill in this art recognize that, in general, singleamino acid substitutions in non-essential regions of a polypeptide donot substantially alter biological activity (see, e.g., Watson et al.Molecular Biology of the Gene, 4th Edition, 1987, The Bejacmin/CummingsPub. co., p. 224). Also included within the definition, is thecatalytically active fragment of an SP, particularly a single chainprotease portion. Conservative amino acid substitutions are made, forexample, in accordance with those set forth in TABLE 1 as follows:

TABLE 1 Original residue Conservative substitution Ala (A) Gly; Ser, AbuArg (R) Lys, orn Asn (N) Gln; His Cys (C) Ser Gln (Q) Asn Glu (E) AspGly (G) Ala; Pro His (H) Asn; Gln Ile (I) Leu; Val; Met; Nle; Nva Leu(L) Ile; Val; Met; Nle; Nv Lys (K) Arg; Gln; Glu Met (M) Leu; Tyr; Ile;NLe Val Ornitine Lys; Arg Phe (F) Met; Leu; Tyr Ser (S) Thr Thr (T) SerTrp (W) Tyr Tyr (Y) Trp; Phe Val (V) Ile; Leu; Met; Nle; NvOther substitutions are also permissible and can be determinedempirically or in accord with known conservative substitutions.

As used herein, Abu is 2-aminobutyric acid; Orn is ornithine.

As used herein, the amino acids, which occur in the various amino acidsequences appearing herein, are identified according to theirwell-known, three-letter or one-letter abbreviations. The nucleotides,which occur in the various DNA fragments, are designated with thestandard single-letter designations used routinely in the art.

As used herein, a probe or primer based on a nucleotide sequencedisclosed herein, includes at least 10, 14, typically at least 16contiguous sequence of nucleotides of SEQ ID No. 5 or SEQ ID No. 22, andprobes of at least 30, 50 or 100 contiguous sequence of nucleotides ofSEQ ID No. 5 or SEQ ID No. 22. The length of the probe or primer forunique hybridization is a function of the complexity of the genome ofinterest.

As used herein, amelioration of the symptoms of a particular disorder byadministration of a particular pharmaceutical composition refers to anylessening, whether permanent or temporary, lasting or transient that canbe attributed to or associated with administration of the composition.

As used herein, antisense polynucleotides refer to synthetic sequencesof nucleotide bases complementary to mRNA or the sense strand ofdouble-stranded DNA. Admixture of sense and antisense polynucleotidesunder appropriate conditions leads to the binding of the two molecules,or hybridization. When these polynucleotides bind to (hybridize with)mRNA, inhibition of protein synthesis (translation) occurs. When thesepolynucleotides bind to double-stranded DNA, inhibition of RNA synthesis(transcription) occurs. The resulting inhibition of translation and/ortranscription leads to an inhibition of the synthesis of the proteinencoded by the sense strand. Antisense nucleic acid molecules typicallycontain a sufficient number of nucleotides to specifically bind to atarget nucleic acid, generally at least 5 contiguous nucleotides, oftenat least 14 or 16 or 30 contiguous nucleotides or modified nucleotidescomplementary to the coding portion of a nucleic acid molecule thatencodes a gene of interest, for example, nucleic acid encoding a singlechain protease domain of an SP.

As used herein, an array refers to a collection of elements, such asantibodies, containing three or more members. An addressable array isone in which the members of the array are identifiable, typically byposition on a solid phase support. Hence, in general the members of thearray are immobilized on discrete identifiable loci on the surface of asolid phase.

As used herein, antibody refers to an immunoglobulin, whether natural orpartially or wholly synthetically produced, including any derivativethereof that retains the specific binding ability the antibody. Henceantibody includes any protein having a binding domain that is homologousor substantially homologous to an immunoglobulin binding domain.Antibodies include members of any immunoglobulin claims, including IgG,IgM, IgA, IgD and IgE.

As used herein, antibody fragment refers to any derivative of anantibody that is less than full-length, retaining at least a portion ofthe full-length antibody's specific binding ability. Examples ofantibody fragments include, but are not limited to, Fab, Fab′, F(ab)₂,single-chain Fvs (scFV), FV, dsFV diabody and Fd fragments. The fragmentcan include multiple chains linked together, such as by disulfidebridges. An antibody fragment generally contains at least about 50 aminoacids and typically at least 200 amino acids.

As used herein, an Fv antibody fragment is composed of one variableheavy domain (V_(H)) and one variable light domain linked by noncovalentinteractions.

As used herein, a dsFV refers to an Fv with an engineered intermoleculardisulfide bond, which stabilizes the V_(H)-V_(L) pair.

As used herein, an F(ab)₂ fragment is an antibody fragment that resultsfrom digestion of an immunoglobulin with pepsin at pH 4.0–4.5; it can berecombinantly expressed to produce the equivalent fragment.

As used herein, Fab fragments are antibody fragments that result fromdigestion of an immunoglobulin with papain; they can be recombinantlyexpressed to produce the equivalent fragment.

As used herein, scFVs refer to antibody fragments that contain avariable light chain (V_(L)) and variable heavy chain (V_(H)) covalentlyconnected by a polypeptide linker in any order. The linker is of alength such that the two variable domains are bridged withoutsubstantial interference. Included linkers are (Gly-Ser)_(n) residueswith some Glu or Lys residues dispersed throughout to increasesolubility.

As used herein, humanized antibodies refer to antibodies that aremodified to include human sequences of amino acids so thatadministration to a human does not provoke an immune response. Methodsfor preparation of such antibodies are known. For example, to producesuch antibodies, the encoding nucleic acid in the hybridoma or otherprokaryotic or eukaryotic cell, such as an E. coli or a CHO cell, thatexpresses the monoclonal antibody is altered by recombinant nucleic acidtechniques to express an antibody in which the amino acid composition ofthe non-variable region is based on human antibodies. Computer programshave been designed to identify such non-variable regions.

As used herein, diabodies are dimeric scFV; diabodies typically haveshorter peptide linkers than scFvs, and they generally dimerize.

As used herein, production by recombinant means by using recombinant DNAmethods means the use of the well known methods of molecular biology forexpressing proteins encoded by cloned DNA.

As used herein the term assessing is intended to include quantitativeand qualitative determination in the sense of obtaining an absolutevalue for the activity of an SP, or a domain thereof, present in thesample, and also of obtaining an index, ratio, percentage, visual orother value indicative of the level of the activity. Assessment can bedirect or indirect and the chemical species actually detected need notof course be the proteolysis product itself but can for example be aderivative thereof or some further substance.

As used herein, biological activity refers to the in vivo activities ofa compound or physiological responses that result upon in vivoadministration of a compound, composition or other mixture. Biologicalactivity, thus, encompasses therapeutic effects and pharmaceuticalactivity of such compounds, compositions and mixtures. Biologicalactivities can be observed in in vitro systems designed to test or usesuch activities. Thus, for purposes herein the biological activity of aluciferase is its oxygenase activity whereby, upon oxidation of asubstrate, light is produced.

As used herein, functional activity refers to a polypeptide or portionthereof that displays one or more activities associated with afull-length protein. Functional activities include, but are not limitedto, biological activity, catalytic or enzymatic activity, antigenicity(ability to bind to or compete with a polypeptide for binding to ananti-polypeptide antibody), immunogenicity, ability to form multimers,the ability to specifically bind to a receptor or ligand for thepolypeptide.

As used herein, a conjugate refers to the compounds provided herein thatinclude one or more SPs, including an MTSP10, particularly single chainprotease domains thereof, and one or more targeting agents. Theseconjugates include those produced by recombinant means as fusionproteins, those produced by chemical means, such as by chemicalcoupling, through, for example, coupling to sulfhydryl groups, and thoseproduced by any other method whereby at least one SP, or a domainthereof, is linked, directly or indirectly via linker(s) to a targetingagent.

As used herein, a targeting agent is any moiety, such as a protein oreffective portion thereof, that provides specific binding of theconjugate to a cell surface receptor, which, can internalize theconjugate or SP portion thereof. A targeting agent can also be one thatpromotes or facilitates, for example, affinity isolation or purificationof the conjugate; attachment of the conjugate to a surface; or detectionof the conjugate or complexes containing the conjugate.

As used herein, an antibody conjugate refers to a conjugate in which thetargeting agent is an antibody.

As used herein, derivative or analog of a molecule refers to a portionderived from or a modified version of the molecule.

As used herein, an effective amount of a compound for treating aparticular disease is an amount that is sufficient to ameliorate, or insome manner reduce the symptoms associated with the disease. Such amountcan be administered as a single dosage or can be administered accordingto a regimen, whereby it is effective. The amount can cure the diseasebut, typically, is administered in order to ameliorate the symptoms ofthe disease. Repeated administration can be required to achieve thedesired amelioration of symptoms.

As used herein equivalent, when referring to two sequences of nucleicacids means that the two sequences in question encode the same sequenceof amino acids or equivalent proteins. When equivalent is used inreferring to two proteins or peptides, it means that the two proteins orpeptides have substantially the same amino acid sequence with only aminoacid substitutions (such as, but not limited to, conservative changessuch as those set forth in Table 1 above) that do not substantiallyalter the activity or function of the protein or peptide. Whenequivalent refers to a property, the property does not need to bepresent to the same extent (e.g., two peptides can exhibit differentrates of the same type of enzymatic activity), but the activities areusually substantially the same. Complementary, when referring to twonucleotide sequences, means that the two sequences of nucleotides arecapable of hybridizing, typically with less than 25%, 15%, 5% or 0%mismatches between opposed nucleotides. If necessary the percentage ofcomplementarity will be specified. Typically the two molecules areselected such that they will hybridize under conditions of highstringency.

As used herein, an agent that modulates the activity of a protein orexpression of a gene or nucleic acid either decreases or increases orotherwise alters the activity of the protein or, in some manner up- ordown-regulates or otherwise alters expression of the nucleic acid in acell.

As used herein, inhibitor of the activity of an SP encompasses anysubstance that prohibits or decrease production, post-translationalmodification(s), maturation, or membrane localization of the SP or anysubstance that interferes with or decreases the proteolytic efficacy ofthereof, particularly of a single chain form in an in vitro screeningassay.

As used herein, a method for treating or preventing neoplastic diseasemeans that any of the symptoms, such as the tumor, metastasis thereof,the vascularization of the tumors or other parameters by which thedisease is characterized are reduced, ameliorated, prevented, placed ina state of remission, or maintained in a state of remission. It alsomeans that the hallmarks of neoplastic disease and metastasis can beeliminated, reduced or prevented by the treatment. Non-limiting examplesof the hallmarks include uncontrolled degradation of the basementmembrane and proximal extracellular matrix, migration, division, andorganization of the endothelial cells into new functioning capillaries,and the persistence of such functioning capillaries.

As used herein, pharmaceutically acceptable salts, esters or otherderivatives of the conjugates include any salts, esters or derivativesthat can be readily prepared by those of skill in this art using knownmethods for such derivatization and that produce compounds that can beadministered to animals or humans without substantial toxic effects andthat either are pharmaceutically active or are prodrugs.

As used herein, a prodrug is a compound that, upon in vivoadministration, is metabolized or otherwise converted to thebiologically, pharmaceutically or therapeutically active form of thecompound. To produce a prodrug, the pharmaceutically active compound ismodified such that the active compound is regenerated by metabolicprocesses. The prodrug can be designed to alter the metabolic stabilityor the transport characteristics of a drug, to mask side effects ortoxicity, to improve the flavor of a drug or to alter othercharacteristics or properties of a drug. By virtue of knowledge ofpharmacodynamic processes and drug metabolism in vivo, those of skill inthis art, once a pharmaceutically active compound is known, can designprodrugs of the compound (see, e.g., Nogrady (1985) Medicinal ChemistryA Biochemical Approach, Oxford University Press, New York, pages388–392).

As used herein, a drug identified by the screening methods providedherein refers to any compound that is a candidate for use as atherapeutic or as a lead compound for the design of a therapeutic. Suchcompounds can be small molecules, including small organic molecules,peptides, peptide mimetics, antisense molecules or dsRNA, such as RNAi,antibodies, fragments of antibodies, recombinant antibodies and othersuch compounds that can serve as drug candidates or lead compounds.

As used herein, a peptidomimetic is a compound that mimics theconformation and certain stereochemical features of the biologicallyactive form of a particular peptide. In general, peptidomimetics aredesigned to mimic certain desirable properties of a compound, but notthe undesirable properties, such as flexibility, that lead to a loss ofa biologically active conformation and bond breakdown. Peptidomimeticsmay be prepared from biologically active compounds by replacing certaingroups or bonds that contribute to the undesirable properties withbioisosteres. Bioisosteres are known to those of skill in the art. Forexample the methylene bioisostere CH₂S has been used as an amidereplacement in enkephalin analogs (see, e.g., Spatola (1983) pp. 267–357in Chemistry and Blochemistry of Amino Acids, Peptides, and Proteins,Weistein, Ed. volume 7, Marcel Dekker, New York). Morphine, which can beadministered orally, is a compound that is a peptidomimetic of thepeptide endorphin. For purposes herein, cyclic peptides are includedamong pepidomimetics.

As used herein, a promoter region or promoter element refers to asegment of DNA or RNA that controls transcription of the DNA or RNA towhich it is operatively linked. The promoter region includes specificsequences that are sufficient for RNA polymerase recognition, bindingand transcription initiation. This portion of the promoter region isreferred to as the promoter. In addition, the promoter region includessequences that modulate this recognition, binding and transcriptioninitiation activity of RNA polymerase. These sequences can be cis actingor can be responsive to trans acting factors. Promoters, depending uponthe nature of the regulation, can be constitutive or regulated.Exemplary promoters contemplated for use in prokaryotes include thebacteriophage T7 and T3 promoters.

As used herein, a receptor refers to a molecule that has an affinity fora given ligand. Receptors can be naturally-occurring or syntheticmolecules. Receptors can also be referred to in the art as anti-ligands.As used herein, the receptor and anti-ligand are interchangeable.Receptors can be used in their unaltered state or as aggregates withother species. Receptors can be attached, covalently or noncovalently,or in physical contact with, to a binding member, either directly orindirectly via a specific binding substance or linker. Examples ofreceptors, include, but are not limited to: antibodies, cell membranereceptors surface receptors and internalizing receptors, monoclonalantibodies and antisera reactive with specific antigenic determinants[such as on viruses, cells, or other materials], drugs, polynucleotides,nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides,cells, cellular membranes, and organelles.

Examples of receptors and applications using such receptors, include butare not restricted to:

a) enzymes: specific transport proteins or enzymes essential to survivalof microorganisms, which could serve as targets for antibiotic [ligand]selection;

b) antibodies: identification of a ligand-binding site on the antibodymolecule that combines with the epitope of an antigen of interest can beinvestigated; determination of a sequence that mimics an antigenicepitope can lead to the development of vaccines of which the immunogenis based on one or more of such sequences or lead to the development ofrelated diagnostic agents or compounds useful in therapeutic treatmentssuch as for auto-immune diseases

c) nucleic acids: identification of ligand, such as protein or RNA,binding sites;

d) catalytic polypeptides: polymers, including polypeptides, that arecapable of promoting a chemical reaction involving the conversion of oneor more reactants to one or more products; such polypeptides generallyinclude a binding site specific for at least one reactant or reactionintermediate and an active functionality proximate to the binding site,in which the functionality is capable of chemically modifying the boundreactant (see, e.g., U.S. Pat. No. 5,215,899);

e) hormone receptors: determination of the ligands that bind with highaffinity to a receptor is useful in the development of hormonereplacement therapies; for example, identification of ligands that bindto such receptors can lead to the development of drugs to control bloodpressure; and

f) opiate receptors: determination of ligands that bind to the opiatereceptors in the brain is useful in the development of less-addictivereplacements for morphine and related drugs.

As used herein, sample refers to anything that contains an analyte forwhich an analyte assay is desired. The sample can be a biologicalsample, such as a biological fluid or a biological tissue. Examples ofbiological fluids include urine, blood, plasma, serum, saliva, semen,stool, sputum, cerebral spinal fluid, tears, mucus, sperm, amnioticfluid or the like. Biological tissues are aggregates of cells, usuallyof a particular kind together with their intercellular substance thatform one of the structural materials of a human, animal, plant,bacterial, fungal or viral structure, including connective, epithelium,muscle and nerve tissues. Examples of biological tissues also includeorgans, tumors, lymph nodes, arteries and individual cell(s).

As used herein: stringency of hybridization in determining percentagemismatch is as follows:

1) high stringency: 0.1×SSPE, 0.1% SDS, 65° C.

2) medium stringency: 0.2×SSPE, 0.1% SDS, 50° C.

3) low stringency: 1.0×SSPE, 0.1% SDS, 50° C.

Those of skill in this art know that the washing step selects for stablehybrids and also know the ingredients of SSPE (see, e.g., Sambrook, E.F. Fritsch, T. Maniatis, in: Molecular Cloning, A Laboratory Manual,Cold Spring Harbor Laboratory Press (1989), vol. 3, p. B. 13, see, also,numerous catalogs that describe commonly used laboratory solutions).SSPE is pH 7.4 phosphate- buffered 0.18 NaCl. Further, those of skill inthe art recognize that the stability of hybrids is determined by T_(m),which is a function of the sodium ion concentration and temperature(T_(m)=81.5° C.−16.6(log₁₀[Na⁺])+0.41 (% G+C)−600/l)), so that the onlyparameters in the wash conditions critical to hybrid stability aresodium ion concentration in the SSPE (or SSC) and temperature.

It is understood that equivalent stringencies can be achieved usingalternative buffers, salts and temperatures. By way of example and notlimitation, procedures using conditions of low stringency are as follows(see also Shilo and Weinberg, Proc. Natl. Acad. Sci. USA 78:6789–6792(1981)): Filters containing DNA are pretreated for 6 hours at 40° C. ina solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmonsperm DNA (10×SSC is 1.5 M sodium chloride, and 0.15 M sodium citrate,adjusted to a pH of 7).

Hybridizations are carried out in the same solution with the followingmodifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon spermDNA, 10% (wt/vol) dextran sulfate, and 5–20×10⁶ cpm ³²P-labeled probe isused. Filters are incubated in hybridization mixture for 18–20 hours at40° C., and then washed for 1.5 hours at 55° C. in a solution containing2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The washsolution is replaced with fresh solution and incubated an additional 1.5hours at 60° C. Filters are blotted dry and exposed for autoradiography.If necessary, filters are washed for a third time at 65–68° C. andreexposed to film. Other conditions of low stringency which can be usedare well known in the art (e.g., as employed for cross-specieshybridizations).

By way of example and not way of limitation, procedures using conditionsof moderate stringency include, for example, but are not limited to,procedures using such conditions of moderate stringency are as follows:Filters containing DNA are pretreated for 6 hours at 55° C. in asolution containing 6×SSC, 5×Denhart's solution, 0.5% SDS and 100 μg/mldenatured salmon sperm DNA. Hybridizations are carried out in the samesolution and 5–20×10⁶ cpm ³²P-labeled probe is used. Filters areincubated in hybridization mixture for 18–20 hours at 55° C., and thenwashed twice for 30 minutes at 60° C. in a solution containing 1×SSC and0.1% SDS. Filters are blotted dry and exposed for autoradiography. Otherconditions of moderate stringency which can be used are well-known inthe art. Washing of filters is done at 37° C. for 1 hour in a solutioncontaining 2×SSC, 0.1% SDS.

By way of example and not way of limitation, procedures using conditionsof high stringency are as follows: Prehybridization of filterscontaining DNA is carried out for 8 hours to overnight at 65° C. inbuffer composed of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP,0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA.Filters are hybridized for 48 hours at 65° C. in prehybridizationmixture containing 100 μg/ml denatured salmon sperm DNA and 5–20×10⁶ cpmof ³²P-labeled probe. Washing of filters is done at 37° C. for 1 hour ina solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA.This is followed by a wash in 0.1×SSC at 50° C. for 45 minutes beforeautoradiography. Other conditions of high stringency which can be usedare well known in the art.

The term substantially identical or substantially homologous or similarvaries with the context as understood by those skilled in the relevantart and generally means at least 60% or 70%, preferably means at least80%, 85% or more preferably at least 90%, and most preferably at least95% identity.

As used herein, substantially identical to a product means sufficientlysimilar so that the property of interest is sufficiently unchanged sothat the substantially identical product can be used in place of theproduct.

As used herein, substantially pure means sufficiently homogeneous toappear free of readily detectable impurities as determined by standardmethods of analysis, such as thin layer chromatography (TLC), gelelectrophoresis and high performance liquid chromatography (HPLC), usedby those of skill in the art to assess such purity, or sufficiently puresuch that further purification would not detectably alter the physicaland chemical properties, such as enzymatic and biological activities, ofthe substance. Methods for purification of the compounds to producesubstantially chemically pure compounds are known to those of skill inthe art. A substantially chemically pure compound can, however, be amixture of stereoisomers or isomers. In such instances, furtherpurification might increase the specific activity of the compound.

As used herein, target cell refers to a cell that expresses an SP invivo.

As used herein, test substance (or test compound) refers to a chemicallydefined compound (e.g., organic molecules, inorganic molecules,organic/inorganic molecules, proteins, peptides, nucleic acids,oligonucleotides, lipids, polysaccharides, saccharides, or hybrids amongthese molecules such as glycoproteins, etc.) or mixtures of compounds(e.g., a library of test compounds, natural extracts or culturesupernatants, etc.) whose effect on an SP, particularly a single chainform that includes the protease domain or a sufficient portion thereoffor activity, as determined by an in vitro method, such as the assaysprovided herein.

As used herein, the terms a therapeutic agent, therapeutic regimen,radioprotectant or chemotherapeutic mean conventional drugs and drugtherapies, including vaccines, which are known to those skilled in theart. Radiotherapeutic agents are well known in the art.

As used herein, treatment means any manner in which the symptoms of acondition, disorder or disease are ameliorated or otherwise beneficiallyaltered. Treatment also encompasses any pharmaceutical use of thecompositions herein.

As used herein, vector (or plasmid) refers to discrete elements that areused to introduce heterologous nucleic acid into cells for eitherexpression or replication thereof. The vectors typically remainepisomal, but can be designed to effect integration of a gene or portionthereof into a chromosome of the genome. Also contemplated are vectorsthat are artificial chromosomes, such as yeast artificial chromosomesand mammalian artificial chromosomes. Selection and use of such vehiclesare well known to those of skill in the art. An expression vectorincludes vectors capable of expressing DNA that is operatively linkedwith regulatory sequences, such as promoter regions, that are capable ofeffecting expression of such DNA fragments. Thus, an expression vectorrefers to a recombinant DNA or RNA construct, such as a plasmid, aphage, recombinant virus or other vector that, upon introduction into anappropriate host cell, results in expression of the cloned DNA.Appropriate expression vectors are well known to those of skill in theart and include those that are replicable in eukaryotic cells and/orprokaryotic cells and those that remain episomal or those whichintegrate into the host cell genome.

As used herein, protein binding sequence refers to a protein or peptidesequence or a portion of other macromolecules that is capable ofspecific binding to protein or peptide sequences generally, to a set ofprotein or peptide sequences or to a particular protein or peptidesequence.

As used herein, epitope tag refers to a short stretch of amino acidresidues corresponding to an epitope to facilitate subsequentbiochemical and immunological analysis of the epitope tagged protein orpeptide. Epitope tagging is achieved by including the sequence of theepitope tag to the protein-encoding sequence in an appropriateexpression vector. Epitope tagged proteins can be affinity purifiedusing highly specific antibodies raised against the tags.

As used herein, metal binding sequence refers to a protein or peptidesequence that is capable of specific binding to metal ions generally, toa set of metal ions or to a particular metal ion.

As used herein, a combination refers to any association between two oramong more items.

As used herein, a composition refers to a any mixture. It can be asolution, a suspension, liquid, powder, a paste, aqueous, non-aqueous orany combination thereof.

As used herein, fluid refers to any composition that can flow. Fluidsthus encompass compositions that are in the form of semi-solids, pastes,solutions, aqueous mixtures, gels, lotions, creams and other suchcompositions.

As used herein, a cellular extract refers to a preparation or fractionwhich is made from a lysed or disrupted cell.

As used herein, an agent is said to be randomly selected when the agentis chosen randomly without considering the specific sequences involvedin the association of a protein alone or with its associated substrates,binding partners, etc. An example of randomly selected agents is the usea chemical library or a peptide combinatorial library, or a growth brothof an organism or conditioned medium.

As used herein, an agent is said to be rationally selected or designedwhen the agent is chosen on a non-random basis which takes into accountthe sequence of the target site and/or its conformation in connectionwith the agent's action. As described in the Examples, there areproposed binding sites for serine protease and (catalytic) sites in theprotein having SEQ ID NO:3 or SEQ ID NO:4. Agents can be rationallyselected or rationally designed by utilizing the peptide sequences thatmake up these sites. For example, a rationally selected peptide agentcan be a peptide whose amino acid sequence is identical to the ATP orcalmodulin binding sites or domains.

For clarity of disclosure, and not by way of limitation, the detaileddescription is divided into the subsections that follow.

B. MTSP10 Polypeptides, Muteins, Derivatives and Analogs Thereof

MTSPs

The MTSPs are a family of transmembrane serine proteases that are foundin mammals and also other species. MTSPs are of interest because theyappear to be expressed and/or activated at different levels in tumorcells from normal cells, or have functional activity that is differentin tumor cells from normal cells, such as by an alteration in asubstrate therefor, or a cofactor or a receptor.

The MTSPs share a number of common structural features including: aproteolytic extracellular C-terminal domain; a transmembrane domain,with a hydrophobic domain near the N-terminus; a short cytoplasmicdomain; and a variable length stem region that may contain additionalmodular domains. The proteolytic domains share sequence homologyincluding conserved His, Asp, and Ser residues necessary for catalyticactivity that are present in conserved motifs. The MTSPs are normallysynthesized as zymogens and can be activated to two-chain forms bycleavage. It is shown herein that a single chain proteolytic domain canfunction in vitro and, hence is useful in in vitro assays foridentifying agents that modulate the activity of members of this family.

For purposes herein, the protease domain of the MTSP does not have toresult from activation cleavage, which produces a two chain activatedproduct, but rather includes single chain polypeptides where theN-terminii include the consensus sequence ↓VVGG, ↓IVGG, ↓VGLL, ↓ILGG,↓IVQG or ↓IVNG ↓IASG or other such motif. Such polypeptides, althoughnot the result of activation cleavage and not two-chain forms, exhibitproteolytic (catalytic) activity. These protease domain polypeptides areused in assays to screen for agents that modulate the activity of theMTSP10.

The MTSP family is a target for therapeutic intervention and also somemembers can serve as diagnostic markers for tumor development, growthand/or progression. As discussed, the members of this family areinvolved in proteolytic processes that are implicated in tumordevelopment, growth and/or progression. This implication is based upontheir functions as proteolytic enzymes in processes related to ECMdegradation and/or remodeling and activation of pro-growth factors,pro-hormones or pro-angiogenic compounds. In addition, their levels ofexpression or level of activation or their apparent activity resultingfrom substrate levels or alterations in substrates and levels thereofdiffers in tumor cells and non-tumor cells in the same tissue. Similarlythe level of co-factors or receptors for these proteases can varybetween tumor and non-tumor cells. Hence, protocols and treatments thatalter their activity, such as their proteolytic activities and roles insignal transduction, and/or their expression, such as by contacting themwith a compound that modulates their activity and/or expression, couldimpact tumor development, growth and/or progression. Also, in someinstances, the level of activation and/or expression can be altered intumors, such as lung carcinoma, colon adenocarcinoma and ovariancarcinoma.

MTSP10

MTSP10 is of interest because it is expressed or is active in tumorcells. The MTSP provided herein can serve as a diagnostic marker forparticular tumors, by virtue of a level of activity and/or expression orfunction in a subject (i.e. a mammal, particularly a human) withneoplastic disease, compared to a subject or subjects that do not havethe neoplastic disease. In addition, detection of activity (and/orexpression) in a particular tissue can be indicative of neoplasticdisease. It is shown herein, that MTSP10s provided herein are expressedand/or activated in certain tumors; hence their activation or expressioncan serve as a diagnostic marker for tumor development, growth and/orprogression. In other instances, the MTSP polypeptide can exhibitaltered activity by virtue of a change in activity or expression of aco-factor, a substrate or a receptor. In addition, in some instances,these MTSPs and/or variants thereof can be shed from cell surfaces.Detection of the shed MTSPs, particularly the extracellular proteasedomains, in body fluids, such as serum, blood, saliva, cerebral spinalfluid, synovial fluid and interstitial fluids, urine, sweat and othersuch fluids and secretions, can serve as a diagnostic tumor marker. Inparticular, detection of higher levels of such shed polypeptides in asubject compared to a subject known not to have any neoplastic diseaseor compared to earlier samples from the same subject, can be indicativeof neoplastic disease in the subject.

Polypeptides and Muteins

Provided herein are isolated substantially pure single chain and twochain polypeptides that contain the protease domain of an MTSP10. Thepolypeptides also can include other non-MTSP sequences of amino acids,but includes the protease domain or a sufficient portion thereof toexhibit catalytic activity in any in vitro assay that assess suchprotease activity, such as any provided herein.

MTSP10 polypeptides provided herein are expressed or activated by or intumor cells, typically at a level that differs from the level in whichthey are expressed by or activated in a non-tumor cell of the same type.Hence, for example, if the MTSP is expressed in an cervical tumor cell,it is expressed or active at a different level from in non-tumorcervical cells. MTPS9 expression or activation can be indicative ofcervial, lung, esophogeal, colon, prostate, uterine, pancreatic, breastand other tumors.

Isolated, substantially pure proteases that include protease domains ora catalytically active portion thereof are provided. Provided are singlechain forms and two chain forms of the MTSP10. The protease domains canbe included in a longer protein, and such longer protein is optionallythe MTSP10 zymogen. Exemplary MTSP10-encoding nucleic acid and proteinsequences of a protease domain are set forth in SEQ ID Nos. 5 and 6.Full-length MTSP10-encoding nucleic acid molecules that contain thesequence set forth in SEQ ID No. 5 or SEQ ID No. 22 and polypeptidesthat include the sequence of amino acids set forth as residues 1–230 inSEQ ID No. 6 or SEQ ID No. 23 or catalytically active portions thereofare also provided herein. Thus, an MTSP10 polypeptide includes thesequence of amino acids set forth as residues 1–230 SEQ ID No. 6 orresidues 463–692 of SEQ ID No. 23 are provided. Smaller portions thereofthat retain protease activity are contemplated.

Substantially purified MTSP10 protease is encoded by a nucleic acid thathybridizes to a nucleic acid molecule containing the protease domainencoded by the sequence of nucleotides that encodes residues 1–230 setforth in SEQ. ID No. 5 under at least moderate, generally high,stringency conditions, such that the protease domain encoding nucleicacid thereof hybridizes along its full-length or at least 70%, 80% or90% of the full-length. In certain embodiments the substantiallypurified MTSP protease is a single chain polypeptide that includessubstantially the sequence of amino acids set forth as residues 1–230 inSEQ ID No. 6 or a catalytically active portion thereof.

Also included are substantially purified MTSP10 zymogens, activated twochain forms, single chain protease domains and two chain proteasedomains. These polypeptides are encoded by a nucleic acid that includessequence encoding a protease domain that exhibits proteolytic activityand that hybridizes to a nucleic acid molecule having a nucleotidesequence set forth in SEQ ID No. 5 or SEQ ID No. 22, typically undermoderate, generally under high stringency, conditions and generallyalong the full-length or along at least about 70%, 80% or 90% of thefull-length (or substantially the full-length) of the protease domain.Splice variants are also contemplated herein.

Structural Features

The catalytic triad of the MTSP10 in SEQ ID No. 23 is H₅₀₃, D₅₅₁ andS₆₄₇. Disulfide bonds pairing in MTSP10 is as follows: C₄₈₈–C₅₀₄,C₅₈₇–C₆₅₃; C₆₁₉–C₆₃₂; C₆₄₃–C₆₇₃. Cys₅₇₃, which is in the proteasedomain, binds to Cys₅₉₆ outside the domain, and is unpaired in thesingle chain form of the protease domain.

MTSP10 includes several domainsin addition to a catalytic domainresidues 463–692 (SEQ ID No. 23; corresponding to residue 1–230 of SEQID No. 6) and a transmembrane domain. These include 2 CUB domains ataa104–217 and aa222–335, respectively; three LDLa domains at aaa340–377, aa381–412 and aa415–453 respectively.

Protease Domains

MTSP protease domains include the single chain protease domains ofMTSP10. Provided are the protease domains or proteins that include aportion of an MTSP that is the protease domain of any MTSP, particularlya MTSP10. The protein can also include other non-MTSP sequences of aminoacids, but includes the protease domain or a sufficient portion thereofto exhibit catalytic activity in any in vitro assay that assess suchprotease activity, such as any provided herein. Also provided are twochain activated forms of the full length protease and also two chainforms of the protease domain. Thus, isolated, substantially pureproteases that include the protease domains or catalytically activeportions thereof as single chain forms of SPs are provided. The proteasedomains can be included in a longer protein, and such longer protein isoptionally the activated MTSP10 protein, up to and including aful-lenght, or an MTSP10 zymogen.

In particular, exemplary protease domains include at least a sufficientportion of sequences of amino acids set forth of SEQ ID No. 6 or SEQ IDNo. 23 (encoded by nucleotides in SEQ ID No. 5 or SEQ ID No. 22) toretain catalytic activity in vitro.

As noted, the protease domains of an MTSP are single-chain polypeptidesor two-chain polypeptides, with an N-terminus (such as IV, VV, IL andII) generated at the cleavage site (generally having the consensussequence R↓VVGG, R↓IVGG, R↓IVQ, R↓IVNG, R↓ILGG, R↓VGLL, R↓ILGG or avariation thereof; an N-terminus R↓V or R↓I, where the arrow representsthe cleavage point) when the zymogen is activated. The protease domainof an exemplary MTSP10, produced is produced by activation cleavagebetween an R462 and the I at residue 463 of SEQ ID No. 23 (R↓I) includesthe sequence R↓IIGGT, as set forth in SEQ ID No. 23. A single chain formincludes residues 1–230 of SEQ ID No. 6 or catalytically activefragments thereof. Hence any length polyeptide that includes theprotease domain (residues 463–692 of SEQ ID No. 23) or catalyticallyactive fragments thereof, is contemplated herein. Two chain forms alsoare provided and include at least a polypeptide from C₂₉₆ up to andincluding C₅₇₃ of SEQ ID No. 23 or corresponding residues of an MTSP10that has at least 60%, 70%, 80%, 90%, or 95% sequence identity therewith and/or is:

-   -   a) a polypeptide that includes the sequence of amino acids set        forth in SEQ ID No. 6, particularly amino acids 1–230;    -   b) a polypeptide that includes the sequence of amino acids set        forth in SEQ ID No. 23;    -   c) a polypeptide encoded by a sequence of nucleotides that        hybridizes under conditions of low, moderate or high stringency        to the sequence of nucleotides set forth in SEQ ID No. 5 or to        SEQ ID No. 22 or hybridizes along at least 70%, 80%, 90% or 95%        of its full-length;    -   d) a polypeptide that includes the sequence of amino acids set        forth in SEQ ID No. 6 or SEQ ID No. 23 or a catalytically active        portion thereof;    -   e) a polypeptide that includes a sequence of amino acids having        at least about 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,        87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or        99% sequence identity with the sequence of amino acids set forth        in SEQ ID No. 6 or SEQ ID No. 23; and/or    -   f) a protease domain of a polypeptide encoded by a splice        variant of a sequence of nucleotides that encodes an MTSP10 of        any of a)–e).

Also provided are polypeptides that are encoded by nucleic acidmolecules that meet criteria specified below as (a)-(e)

Muteins and Derivatives

Full-length MTSP10, zymogen and activated forms thereof and MTSP10protease domains, portions thereof, and muteins and derivatives of suchpolypeptides are provided. The domains, fragments, derivatives oranalogs of an MTSP10 that are functionally active are capable ofexhibiting one or more functional activities associated with the MTSP10polypeptide, such as serine protease activity, immunogenicity andantigenicity, are provided.

Among the derivatives are those based on animal MTSP10s, including, butare not limited to, rodent, such as mouse and rat; fowl, such aschicken; ruminants, such as goats, cows, deer, sheep; ovine, such aspigs; and humans. For example, MTSP10 derivatives can be made byaltering their sequences by substitutions, additions or deletions.MTSP10 derivatives include, but are not limited to, those containing, asa primary amino acid sequence, all or part of the amino acid sequence ofMTSP10, including altered sequences in which functionally equivalentamino acid residues are substituted for residues within the sequenceresulting in a silent change. For example, one or more amino acidresidues within the sequence can be substituted by another amino acid ofa similar polarity which acts as a functional equivalent, resulting in asilent alteration. Substitutes for an amino acid within the sequence canbe selected from other members of the class to which the amino acidbelongs. For example, the nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophanand methionine. The polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine. The positivelycharged (basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid (see, e.g., Table 1). Muteins of the MTSP10 or a domainthereof, such as a protease domain, in which up to about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% of the amino acids are replacedwith another amino acid are provided. Generally such muteins retain atleast about 1%, 2%, 3%, 5%, 7%, 8%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80% or 90% of the protease activity the unmutated protein. Those ofskill in the art recognize that a polyepeptide that retains at least 1%of the activity of the wild-type protease is sufficiently active for usein screening assays or for other applications. Muteins of the MTSP10 ora domain thereof, such as a protease domain, in which up to about 10%,20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of theamino acids are replaced with another amino acid are provided. Generallysuch muteins retain at least about 1%, 2%, 3%, 5%, 7%, 8%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80% or 90% (in increased activity, i.e., 101,102, 103, 104, 105, 110% or greater) of the protease activity theunmutated protein.

Included among the polypeptides provided herein are the MTSP10 proteasedomain or a polypeptide with amino acid changes such that thespecificity and protease activity remains substantially unchanged orchanged (increased or decreased) by a specified percentage, such as 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.5%. In particular, a substantially purifiedmammalian MTSP polypeptide is provided that has a transmembrane (TM)domain and additionally includes two CUB domains, three LDL receptortype a domains and a serine protease catalytic domain is provided.

Also provided is a substantially purified protein containing a sequenceof amino acids that has at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identity to the MTSP10 where the percentage identity is determinedusing standard algorithms and gap penalties that maximize the percentageidentity. The human MTSP10 polypeptide is included, although othermammalian MTSP10 polypeptides are contemplated. The precise percentageof identity can be specified if needed.

Muteins in which one or more of the Cys residues, particularly, aresidue that is paired in the activated two form, but unpaired in theprotease domain alone is/are replaced with any amino acid, typically,although not necessarily, a conservative amino acid residue, such asSer, are contemplated. Muteins of MTSP10, particularly those in whichCys residues, such as the Cys₅₇₃ in the single chain protease domain, isreplaced with another amino acid, such as Ser, Gly or Ala, that does noteliminate the activity, are provided. Also provided are substantiallypurified MTSP10 polypeptides and functional domains thereof, includingcatalytically active domains and portions, that have at least about 60%,70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with aprotease domain that includes a sequence of amino acids set forth in SEQID No. 6, particularly amino acids 1–230 or catalytically activefragments thereof.

Muteins of the protein are also provided in which amino acids arereplaced with other amino acids. Among the muteins are those in whichthe Cys residues, is/are replaced typically with a conservative aminoacid residues, such as a serine. Such muteins are also provided herein.Muteins in which 10%, 20%, 30%, 35%, 40%, 45%, 50% or more of the aminoacids are replaced but the resulting polypeptide retains at least about1%, 2%, 3%, 5%, 7%, 8%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%,80%, 90% or 95% of the catalytic activity as the unmodified form for thesame substrate.

Muteins can be made by making conservative amino acid substitions andalso non-conservative amino acid substitutions. For example, amino acidsubstitutions the desirably alter properties of the proteins can bemade. In one embodiment, mutations that prevent degradation of thepolypeptide can be made. Many proteases cleave after basic residues,such as R and K; to eliminate such cleavage, the basic residue isreplaced with a non-basic residue. Also, non-conservative changes atamino acids outside of the protease domain can be effected withoutaltering protease activity. Non-conservative changes at amino acids thatare responsible for activities other than protease activity may bedesirable. For example, interaction of the protease with an inhibitorcan be blocked while retaining catalytic activity by effecting anon-conservative change at the site interaction of the inhibitor withthe protease. Similarly, receptor binding can be altered withoutaltering catalytic activity by effecting a non-conservative orconservative at a site of interaction of the receptor with the protease.

Antigenic epitopes that contain at least 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 20, 25, 30, 40, 50, and typically 10–15 amino acids of theMTSP10 polypeptide are provided. These antigenic epitopes are used, forexample, to raise antibodies. Antibodies specific for each epitope orcombinations thereof and for single and two-chain forms are alsoprovided.

Nucleic Acid Molecules, Vectors and Plasmids, Cells and Expression ofMTSP10 Polyeptides

Nucleic Acid Molecules

Due to the degeneracy of nucleotide coding sequences, other nucleicsequences which encode substantially the same amino acid sequence as aMTSP are contemplated. These include but are not limited to nucleic acidmolecules that include all or portions of MTSP10-encoding genes that arealtered by the substitution of different codons that encode the aminoacid residue within the sequence, thus producing a silent change.

Nucleic Acids

Also provided herein are nucleic acid molecules that encode MTSP10polypeptides and the encoded proteins. In particular, nucleic acidmolecules encoding MTSP10 from animals, including splice variantsthereof are provided. The encoded proteins are also provided. Alsoprovided are functional domains thereof. For each of the nucleic acidmolecules provided, the nucleic acid can be DNA or RNA or PNA or othernucleic acid analogs or can include non-natural nucleotide bases. Alsoprovided are isolated nucleic acid molecules that include a sequence ofnucleotides complementary to the nucleotide sequence encoding an MTSP.

Also provided are nucleic acid molecules that encode single chain or twochain MTSP proteases that have proteolytic activity in an in vitroproteolysis assay and that have at least 60%, 70%, 75%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% sequence identity with the full-length of a proteasedomain of an MTSP10 polypeptide, or that hybridize along theirfull-length or along at least about 70%, 80% or 90% of the full-lengthnucleic acid to a nucleic acids that encode a protease domain,particularly under conditions of moderate, generally high, stringency.As above, the encoded polypeptides contain the protease as a singlechain; activated forms thereof can be produced and are provided.

In one embodiment, a nucleic acid molecule that encodes an MTSP,designated MTSP10 is provided. The nucleic acid molecule includes theopen reading frame in the sequence of nucleotides set forth in SEQ IDNo. 5 or SEQ ID No. 23. Also provided are nucleic acid molecules thathybridize under conditions of at least low stringency, moderatestringency, and generally high stringency to the following sequence ofnucleic acids (SEQ ID No. 5) particularly to the open reading frameencompassed by nucleotides that encode a single protease domain thereof,or any domain of MTSP10.

In certain embodiments, the isolated nucleic acid fragment hybridizes tothe nucleic acid having the nucleotide sequence set forth in SEQ ID No.5 under high stringency conditions, and generally contains the sequenceof nucleotides set forth as nucleotides 1–690 in SEQ ID No. 5. Theprotein contains a transmembrane domain (TM) and a serine proteasedomain and can contain additional domains, including a CUB domain andLDLR domain.

Also provided, are muteins of the nucleic acid molecules that encodepolypeptides in which amino acids are replaced with other amino acids.Among the muteins are those in which the Cys residue-encoding codons,is/are replaced with other amino acid residues, such as a codon encodinga serine. Such muteins are also provided herein. Each of such domains isprovided herein as are nucleic acid molecules that include sequences ofnucleotides encoding such domains. Some MTSPs can additionally include aLDLR domain, a scavenger-receptor cysteine rich (SRCR) domain and otherdomains.

The isolated nucleic acid fragment is DNA, including genomic or cDNA, oris RNA, or can include other components, such as peptide nucleic acid(PNA) and other nucleotide analogs. The isolated nucleic acid caninclude additional components, such as heterologous or native promoters,and other transcriptional and translational regulatory sequences, thesegenes can be linked to other genes, such as reporter genes or otherindicator genes or genes that encode indicators.

Also provided are nucleic acid molecules that hybridize to theabove-noted sequences of nucleotides encoding MTSP10 at least at lowstringency, moderate stringency, and typically at high stringency, andthat encode the protease domain and/or the full-length protein or atleast 60%, 70%, 80% or 90% of the full-length protease domain or otherdomains of an MTSP10 or a splice variant or allelic variant thereof.Generally the molecules hybridize under such conditions along theirfull-length or along at least 70%, 80% or 90% of the full-length for atleast one domain and encode at least one domain, such as the protease orextracellular domain, of the polypeptide. In particular, such nucleicacid molecules include any isolated nucleic fragment that encodes atleast one domain of a membrane serine protease, that (1) contains asequence of nucleotides that encodes the protease or a domain thereof,and (2) is selected from among:

-   -   (a) a sequence of nucleotides that encodes the protease or a        domain thereof that includes a sequence of nucleotides set forth        in SEQ ID No. 5, particularly nucleotides 1–690, or SEQ ID No.        22;    -   (b) a sequence of nucleotides that encodes such portion or the        full-length protease and hybridizes under conditions of high        stringency, generally to nucleic acid that is complementary to a        mRNA transcript present in a mammalian cell that encodes such        protein or fragment thereof;    -   (a) a sequence of nucleotides that encodes the protease or a        domain thereof that includes a sequence of nucleotides set        having at least about 60%, 70%, 80%, 90% or 95% sequence        identity the the sequence set forth in SEQ ID No. 5, particulary        nucleotides 1–690, or in SEQ ID No. 22;    -   (c) a sequence of nucleotides that encodes a transmembrane        protease or domain thereof that includes a sequence of amino        acids encoded by such portion or the full-length open reading        frame;    -   (d) a sequence of nucleotides that encodes the protease or a        domain thereof that includes a sequence of nucleotides set        having at least about 60%, 70%, 80%, 90% or 95% sequence        identity the the sequence set forth in SEQ ID No. 5 or SEQ ID        No. 22; and    -   (e) a sequence of nucleotides that encodes the transmembrane        protease that includes a sequence of amino acids encoded by a        sequence of nucleotides that encodes such subunit and hybridizes        under conditions of low, moderate or high stringency to DNA that        is complementary to the mRNA transcript.

The isolated nucleic acids can contain least 10 nucleotides, 25nucleotides, 50 nucleotides, 100 nucleotides, 150 nucleotides, or 200nucleotides or more- contiguous nucleotides of an MTSP10-encodingsequence, or a full-length SP coding sequence. In another embodiment,the nucleic acids are smaller than 35, 200 or 500 nucleotides in length.Nucleic acids that hybridize to or are complementary to anMTSP10-encoding nucleic acid molecule can be single or double-stranded.For example, nucleic acids are provided that include a sequencecomplementary to (specifically are the inverse complement of) at least10, 25, 50, 100, or 200 nucleotides or the entire coding region of anMTSP10 encoding nucleic acid, particularly the protease domain thereof.For MTSP10 the full-length protein or a domain or active fragmentthereof is also provided.

Probes, Primers, Antisense Oligonucleotides and dsRNA

Also provided are fragments thereof that can be used as probes orprimers and that contain at least about 10 nucleotides, 14 nucleotides,generally at least about 16 nucleotides, often at least about 30nucleotides. The length of the probe or primer is a function of the sizeof the genome probed; the larger the genome, the longer the probe orprimer required for specific hybridization to a single site. Those ofskill in the art can select appropriately sized probes and primers.Generally probes and primers as described are single-stranded. Doublestranded probes and primers can be used, if they are denatured whenused.

Probes and primers derived from the nucleic acid molecules are provided.Such probes and primers contain at least 8, 14, 16, 30, 100 or morecontiguous nucleotides with identity to contiguous nucleotides of anMTSP10, and probes of at least 14, 16, 30, 50 or 100 contiguous sequenceof nucleotides of SEQ ID No. 5 or SEQ ID No. 22. The probes and primersare optionally labelled with a detectable label, such as a radiolabel ora fluorescent tag, or can be mass differentiated for detection by massspectrometry or other means.

Also provided is an isolated nucleic acid molecule that includes thesequence of molecules that is complementary to the nucleotide sequenceencoding MTSP10 or the portion thereof. Double-stranded RNA (dsRNA),such as RNAi is also provided.

Plasmids, Vectors and Cells

Plasmids and vectors containing the nucleic acid molecules are alsoprovided. Cells containing the vectors, including cells that express theencoded proteins are provided. The cell can be a bacterial cell, a yeastcell, a fungal cell, a plant cell, an insect cell or an animal cell.Methods for producing an MTSP or single chain form of the proteasedomain thereof by, for example, growing the cell under conditionswhereby the encoded MTSP is expressed by the cell, and recovering theexpressed protein, are provided herein. As noted, for MTSP10, thefull-length zymogens and activated proteins and activated (two chain)protease and single chain protease domains are provided. As describedherein, the cells are used for expression of the protein, which can besecreted or expressed in the cytoplasm.

As discussed below, the MTSP10 polypeptide, and catalytically activeportions thereof, can be expressed on the surface of a cell. Inaddition, all or portions thereof can be expressed as a secreted proteinusing the native signal sequence or a heterologous signal.Alternatively, all or portions of the polhpeptide can be expressed asinclusion bodies in the cytoplasm and isolated therefrom. The resultingprotein can be treated to refold if necessary.

The above discussion provides an overview and some details of theexemplified MTSP10s.

C. Tumor Specificity and Tissue Expression Profiles

MTSPs are of interest because they appear to be expressed and/oractivated at different levels in tumor cells from normal cells, or havefunctional activity that is different in tumor cells from normal cells,such as by an alteration in a substrate for the MTSP, or a cofactor orreceptor of the MTSP. MTSP10 is of interest because it is expressed oris active in tumor cells. Hence the MTSPs provided herein can serve asdiagnostic markers for certain tumors.

Each MTSP has a characteristic tissue expression profile; the MTSPs inparticular, although not exclusively expressed or activated in tumors,exhibit characteristic tumor tissue expression or activation profiles.In some instances, MTSPs can have different activity in a tumor cellfrom a non-tumor cell by virtue of a change in a substrate or cofactoror receptor therefor or other factor that would alter the functionalactivity of the MTSP. Hence each can serve as a diagnostic marker forparticular tumors, by virtue of a level of activity and/or expression orfunction in a subject (i.e. a mammal, particularly a human) withneoplastic disease, compared to a subject or subjects that do not havethe neoplastic disease. In addition, detection of activity (and/orexpression) in a particular tissue can be indicative of neoplasticdisease. Shed MTSPs in body fluids can be indicative of neoplasticdisease. Also, by virtue of the activity and/or expression profiles ofeach, they can serve as therapeutic targets, such as by administrationof modulators of the activity thereof, or, as by administration of aprodrug specifically activated by one of the MTSPs.

Tissue Expression Profiles

MTSP10

MTSP10 transcript was detected in pancreas, lung and kidney. MTSP10transcript was also detected in small intestine Marathon-Ready cDNA(Clontech). The MTSP10 transcript was detected in breast carcinoma(GI-101), lung carcinoma (LX-1 and GI-117), ovarian carcinoma (GI-102),and pancreatic adenocarcinoma (GI-103). The MTSP10 transcript was weaklydetected in prostatic adenocarcinoma (PC3). The MTSP10 transcript wasalso detected in the CWR22R prostate tumor grown on nude mice. Noapparent signal was detected in two forms of colon adenocarcinomas(GI-112 and CX-1).

D. Identification and Isolation of MTSP10 Polypeptide Genes

The MTSP polypeptides and/or domains thereof, can be obtained by methodswell known in the art for protein purification and recombinant proteinexpression. Any method known to those of skill in the art foridentification of nucleic acids that encode desired genes can be used.Any method available in the art can be used to obtain a full-length(i.e., encompassing the entire coding region) cDNA or genomic DNA cloneencoding an MTSP polypeptide. For example, the polymerase chain reaction(PCR) can be used to amplify a sequence that is expressed in normal andtumor cells or tissues, e.g., nucleic acids encoding an MTSP10polypeptide (SEQ. Nos: 5 and 17), in a genomic or cDNA library.Oligonucleotide primers that hybridize to sequences at the 3′ and 5′termini of the identified sequences can be used as primers to amplify byPCR sequences from a nucleic acid sample (RNA or DNA), generally a cDNAlibrary, from an appropriate source (e.g., tumor or cancer tissue).

PCR can be carried out, e.g., by use of a Perkin-Elmer Cetus thermalcycler and Taq polymerase (Gene Amp™). The DNA being amplified caninclude mRNA or cDNA or genomic DNA from any eukaryotic species. One canchoose to synthesize several different degenerate primers, for use inthe PCR reactions. It is also possible to vary the stringency ofhybridization conditions used in priming the PCR reactions, to amplifynucleic acid homologs (e.g., to obtain MTSP polypeptide sequences fromspecies other than humans or to obtain human sequences with homology toMTSP10 polypeptide) by allowing for greater or lesser degrees ofnucleotide sequence similarity between the known nucleotide sequence andthe nucleic acid homolog being isolated. For cross-specieshybridization, low stringency to moderate stringency conditions areused. For same species hybridization, moderately stringent to highlystringent conditions are used. The conditions can be empiricallydetermined.

After successful amplification of the nucleic acid containing all or aportion of the identified MTSP polypeptide sequence or of a nucleic acidencoding all or a portion of an MTSP polypeptide homolog, that segmentcan be molecularly cloned and sequenced, and used as a probe to isolatea complete cDNA or genomic clone. This, in turn, permits thedetermination of the gene's complete nucleotide sequence, the analysisof its expression, and the production of its protein product forfunctional analysis. Once the nucleotide sequence is determined, an openreading frame encoding the MTSP polypeptide gene protein product can bedetermined by any method well known in the art for determining openreading frames, for example, using publicly available computer programsfor nucleotide sequence analysis. Once an open reading frame is defined,it is routine to determine the amino acid sequence of the proteinencoded by the open reading frame. In this way, the nucleotide sequencesof the entire MTSP polypeptide genes as well as the amino acid sequencesof MTSP polypeptide proteins and analogs can be identified.

Any eukaryotic cell potentially can serve as the nucleic acid source forthe molecular cloning of the MTSP polypeptide gene. The nucleic acidscan be isolated from vertebrate, mammalian, human, porcine, bovine,feline, avian, equine, canine, as well as additional primate sources,insects, plants and other organisms. The DNA can be obtained by standardprocedures known in the art from cloned DNA (e.g., a DNA “library”), bychemical synthesis, by cDNA cloning, or by the cloning of genomic DNA,or fragments thereof, purified from the desired cell (see, e.g.,Sambrook et al. (2001) Molecular Cloning, A Laboratory Manual, 3d Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Glover,D. M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd.,Oxford, U.K. Vol. I, II). Clones derived from genomic DNA can containregulatory and intron DNA regions in addition to coding regions; clonesderived from cDNA will contain only exon sequences. For any source, thegene is cloned into a suitable vector for propagation thereof.

In the molecular cloning of the gene from genomic DNA, DNA fragments aregenerated, some of which will encode the desired gene. The DNA can becleaved at specific sites using various restriction enzymes.Alternatively, one can use DNAse in the presence of manganese tofragment the DNA, or the DNA can be physically sheared, for example, bysonication. The linear DNA fragments then can be separated according tosize by standard techniques, including but not limited to, agarose andpolyacrylamide gel electrophoresis and column chromatography.

Once the DNA fragments are generated, identification of the specific DNAfragment containing the desired gene can be accomplished in a number ofways. For example, a portion of the MTSP polypeptide (of any species)gene (e.g., a PCR amplification product obtained as described above oran oligonucleotide having a sequence of a portion of the knownnucleotide sequence) or its specific RNA, or a fragment thereof bepurified and labeled, and the generated DNA fragments can be screened bynucleic acid hybridization to the labeled probe (Benton and Davis,Science 196:180 (1977); Grunstein and Hogness, Proc. Natl. Acad. Sci.U.S.A. 72:3961 (1975)). Those DNA fragments with substantial homology tothe probe will hybridize. It is also possible to identify theappropriate fragment by restriction enzyme digestion(s) and comparisonof fragment sizes with those expected according to a known restrictionmap if such is available or by DNA sequence analysis and comparison tothe known nucleotide sequence of MTSP polypeptide. Further selection canbe carried out on the basis of the properties of the gene.Alternatively, the presence of the gene can be detected by assays basedon the physical, chemical, or immunological properties of its expressedproduct. For example, cDNA clones, or DNA clones which hybrid-select theproper mRNA, can be selected which produce a protein that, e.g., hassimilar or identical electrophoretic migration, isolectric focusingbehavior, proteolytic digestion maps, antigenic properties, serineprotease activity. If an anti-MTSP polypeptide antibody is available,the protein can be identified by binding of labeled antibody to theputatively MTSP polypeptide synthesizing clones, in an ELISA(enzyme-linked immunosorbent assay)-type procedure.

Alternatives to isolating the MTSP10 polypeptide genomic DNA include,but are not limited to, chemically synthesizing the gene sequence from aknown sequence or making cDNA to the mRNA that encodes the MTSPpolypeptide. For example, RNA for cDNA cloning of the MTSP polypeptidegene can be isolated from cells expressing the protein. The identifiedand isolated nucleic acids then can be inserted into an appropriatecloning vector. A large number of vector-host systems known in the artcan be used. Possible vectors include, but are not limited to, plasmidsor modified viruses, but the vector system must be compatible with thehost cell used. Such vectors include, but are not limited to,bacteriophages such as lambda derivatives, or plasmids such as pBR322 orpUC plasmid derivatives or the Bluescript vector (Stratagene, La Jolla,Calif.). The insertion into a cloning vector can, for example, beaccomplished by ligating the DNA fragment into a cloning vector whichhas complementary cohesive termini. If the complementary restrictionsites used to fragment the DNA are not present in the cloning vector,the ends of the DNA molecules can be enzymatically modified.Alternatively, any site desired can be produced by ligating nucleotidesequences (linkers) onto the DNA termini; these ligated linkers caninclude specific chemically synthesized oligonucleotides encodingrestriction endonuclease recognition sequences. In an alternativemethod, the cleaved vector and MTSP polypeptide gene can be modified byhomopolymeric tailing. Recombinant molecules can be introduced into hostcells via transformation, transfection, infection, electroporation,calcium precipitation and other methods, so that many copies of the genesequence are generated.

In specific embodiments, transformation of host cells with recombinantDNA molecules that incorporate the isolated MTSP polypeptide gene, cDNA,or synthesized DNA sequence enables generation of multiple copies of thegene. Thus, the gene can be obtained in large quantities by growingtransformants, isolating the recombinant DNA molecules from thetransformants and, when necessary, retrieving the inserted gene from theisolated recombinant DNA.

E. Vectors, Plasmids and Cells that Contain Nucleic Acids Encoding anMTSP Polypeptide or Protease Domain Thereof and Expression of MTSPPolypeptides

Vectors and Cells

For recombinant expression of one or more of the MTSP polypeptides, thenucleic acid containing all or a portion of the nucleotide sequenceencoding the MTSP polypeptide can be inserted into an appropriateexpression vector, i.e., a vector that contains the necessary elementsfor the transcription and translation of the inserted protein codingsequence. The necessary transcriptional and translational signals canalso be supplied by the native promoter for MTSP genes, and/or theirflanking regions.

Also provided are vectors that contain nucleic acid encoding the MTSPs.Cells containing the vectors are also provided. The cells includeeukaryotic and prokaryotic cells, and the vectors are any suitable foruse therein.

Prokaryotic and eukaryotic cells, including endothelial cells,containing the vectors are provided. Such cells include bacterial cells,yeast cells, fungal cells, plant cells, insect cells and animal cells.The cells are used to produce an MTSP polypeptide or protease domainthereof by (a) growing the above-described cells under conditionswhereby the encoded MTSP polypeptide or protease domain of the MTSPpolypeptide is expressed by the cell, and then (b) recovering theexpressed protease domain protein. In the exemplified embodiments, theprotease domain is secreted into the medium.

In one embodiment, vectors that include a sequence of nucleotides thatencode a polypeptide that has protease activity and contains all or aportion of only the protease domain, or multiple copies thereof, of anSP protein are provided. Also provided are vectors that include asequence of nucleotides that encodes the protease domain and additionalportions of an SP protein up to and including a full length SP protein,as well as multiple copies thereof. The vectors can be selected forexpression of the SP protein or protease domain thereof in the cell orsuch that the SP protein is expressed as a secreted protein.Alternatively, the vectors can include signals necessary for secretionof encoded proteins. When the protease domain is expressed the nucleicacid is linked to nucleic acid encoding a secretion signal, such as theSaccharomyces cerevisiae α mating factor signal sequence or a portionthereof, or the native signal sequence.

A variety of host-vector systems can be used to express the proteincoding sequence. These include but are not limited to mammalian cellsystems infected with virus (e.g. vaccinia virus, adenovirus, etc.);insect cell systems infected with virus (e.g. baculovirus);microorganisms such as yeast containing yeast vectors; or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system used, any one of anumber of suitable transcription and translation elements can be used.

Any methods known to those of skill in the art for the insertion ofnucleic acid fragments into a vector can be used to construct expressionvectors containing a chimeric gene containing appropriatetranscriptional/translational control signals and protein codingsequences. These methods can include in vitro recombinant DNA andsynthetic techniques and in vivo recombinants (genetic recombination).Expression of nucleic acid sequences encoding MTSP polypeptide, ordomains, derivatives, fragments or homologs thereof, can be regulated bya second nucleic acid sequence so that the genes or fragments thereofare expressed in a host transformed with the recombinant DNAmolecule(s). For example, expression of the proteins can be controlledby any promoter/enhancer known in the art. In a specific embodiment, thepromoter is not native to the genes for MTSP polypeptide. Promoterswhich can be used include but are not limited to the SV40 early promoter(Bernoist and Chambon, Nature 290:304–310 (1981)), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamotoet al., Cell 22:787–797 (1980)), the herpes thymidine kinase promoter(Wagner et al., Proc. Natl. Acad. Sci. USA 78:1441–1445 (1981)), theregulatory sequences of the metallothionein gene (Brinster et al.,Nature 296:39–42 (1982)); prokaryotic expression vectors such as theβ-lactamase promoter (Villa-Kamaroff et al., Proc. Natl. Acad. Sci. USA75:3727–3731 1978)) or the tac promoter (DeBoer et al., Proc. Natl.Acad. Sci. USA 80:21–25 (1983)); see also “Useful Proteins fromRecombinant Bacteria”: in Scientific American 242:79–94 (1980)); plantexpression vectors containing the nopaline synthetase promoter(Herrar-Estrella et al., Nature 303:209–213 (1984)) or the cauliflowermosaic virus 35S RNA promoter (Garder et al., Nucleic Acids Res. 9:2871(1981)), and the promoter of the photosynthetic enzyme ribulosebisphosphate carboxylase (Herrera-Estrella et al., Nature 310:115–120(1984)); promoter elements from yeast and other fungi such as the Gal4promoter, the alcohol dehydrogenase promoter, the phosphoglycerol kinasepromoter, the alkaline phosphatase promoter, and the following animaltranscriptional control regions that exhibit tissue specificity and havebeen used in transgenic animals: elastase I gene control region which isactive in pancreatic acinar cells (Swift et al., Cell 38:639–646 (1984);Ornitz et al., Cold Spring Harbor Symp. Quant. Biol. 50:399–409 (1986);MacDonald, Hepatology 7:425–515 (1987)); insulin gene control regionwhich is active in pancreatic beta cells (Hanahan et al., Nature315:115–122 (1985)), immunoglobulin gene control region which is activein lymphoid cells (Grosschedl et al., Cell 38:647–658 (1984); Adams etal., Nature 318:533–538 (1985); Alexander et al., Mol. Cell Biol.7:1436–1444 (1987)), mouse mammary tumor virus control region which isactive in testicular, breast, lymphoid and mast cells (Leder et al.,Cell 45:485–495 (1986)), albumin gene control region which is active inliver (Pinckert et al., Genes and Devel. 1:268–276 (1987)),alpha-fetoprotein gene control region which is active in liver (Krumlaufet al., Mol. Cell. Biol. 5:1639–1648 (1985); Hammer et al., Science235:53–58 1987)), alpha-1 antitrypsin gene control region which isactive in liver (Kelsey et al., Genes and Devel. 1:161–171 (1987)), betaglobin gene control region which is active in myeloid cells (Mogram etal., Nature 315:338–340 (1985); Kollias et al., Cell 46:89–94 (1986)),myelin basic protein gene control region which is active inoligodendrocyte cells of the brain (Readhead et al., Cell 48:703–712(1987)), myosin light chain-2 gene control region which is active inskeletal muscle (Sani, Nature 314:283–286 (1985)), and gonadotrophicreleasing hormone gene control region which is active in gonadotrophs ofthe hypothalamus (Mason et al., Science 234:1372–1378 (1986)).

In a specific embodiment, a vector is used that contains a promoteroperably linked to nucleic acids encoding an MTSP polypeptide, or adomain, fragment, derivative or homolog, thereof, one or more origins ofreplication, and optionally, one or more selectable markers (e.g., anantibiotic resistance gene). Expression vectors containing the codingsequences, or portions thereof, of an MTSP polypeptide, is made, forexample, by subcloning the coding portions into the EcoRI restrictionsite of each of the three pGEX vectors (glutathione S-transferaseexpression vectors (Smith and Johnson, Gene 7:31–40 (1988)). This allowsfor the expression of products in the correct reading frame. Exemplaryvectors and systems for expression of the protease domains of the MTSPpolypeptides include the well-known Pichia vectors (available, forexample, from Invitrogen, San Diego, Calif.), particularly thosedesigned for secretion of the encoded proteins. The protein can also beexpressed cytoplasmically, such as in the inclusion bodies. Oneexemplary vector is described in the EXAMPLES.

Plasmids for transformation of E. coli cells, include, for example, thepET expression vectors (see, U.S. Pat. No. 4,952,496; available fromNOVAGEN, Madison, Wis.; see, also literature published by Novagendescribing the system). Such plasmids include pET 11a, which containsthe T7lac promoter, T7 terminator, the inducible E. coli lac operator,and the lac repressor gene; pET 12a–c, which contains the T7 promoter,T7 terminator, and the E. coli ompT secretion signal; and pET 15b andpET19b (NOVAGEN, Madison, Wis.), which contain a His-Tag™ leadersequence for use in purification with a His column and a thrombincleavage site that permits cleavage following purification over thecolumn; the T7-lac promoter region and the T7 terminator.

The vectors are introduced into host cells, such as Pichia cells andbacterial cells, such as E. coli, and the proteins expressed therein.Exemplary Pichia strains, include, for example, GS115. Exemplarybacterial hosts contain chromosomal copies of DNA encoding T7 RNApolymerase operably linked to an inducible promoter, such as the lacUVpromoter (see, U.S. Pat. No. 4,952,496). Such hosts include, but are notlimited to, the lysogenic E. coli strain BL21 (DE3).

Expression and Production of Proteins

The MTSP domains, derivatives and analogs can be produced by variousmethods known in the art. For example, once a recombinant cellexpressing an MTSP polypeptide, or a domain, fragment or derivativethereof, is identified, the individual gene product can be isolated andanalyzed. This is achieved by assays based on the physical and/orfunctional properties of the protein, including, but not limited to,radioactive labeling of the product followed by analysis by gelelectrophoresis, immunoassay, cross-linking to marker-labeled product,and assays of proteolytic activity.

The MTSP polypeptides can be isolated and purified by standard methodsknown in the art (either from natural sources or recombinant host cellsexpressing the complexes or proteins), including but not restricted tocolumn chromatography (e.g., ion exchange, affinity, gel exclusion,reversed-phase high pressure and fast protein liquid), differentialcentrifugation, differential solubility, or by any other standardtechnique used for the purification of proteins. Functional propertiescan be evaluated using any suitable assay known in the art.

Alternatively, once an MTSP polypeptide or its domain or derivative isidentified, the amino acid sequence of the protein can be deduced fromthe nucleotide sequence of the gene which encodes it. As a result, theprotein or its domain or derivative can be synthesized by standardchemical methods known in the art (e.g. see Hunkapiller et al, Nature310:105–1 11 (1984)).

Manipulations of MTSP polypeptide sequences can be made at the proteinlevel. Also contemplated herein are MTSP polypeptide proteins, domainsthereof, derivatives or analogs or fragments thereof, which aredifferentially modified during or after translation, e.g., byglycosylation, acetylation, phosphorylation, amidation, derivatizationby known protecting/blocking groups, proteolytic cleavage, linkage to anantibody molecule or other cellular ligand. Any of numerous chemicalmodifications can be carried out by known techniques, including but notlimited to specific chemical cleavage by cyanogen bromide, trypsin,chymotrypsin, papain, V8 protease, NaBH₄, acetylation, formulation,oxidation, reduction, metabolic synthesis in the presence of tunicamycinand other such agents.

In addition, domains, analogs and derivatives of an MTSP polypeptide canbe chemically synthesized. For example, a peptide corresponding to aportion of an MTSP polypeptide, which includes the desired domain orwhich mediates the desired activity in vitro can be synthesized by useof a peptide synthesizer. Furthermore, if desired, nonclassical aminoacids or chemical amino acid analogs can be introduced as a substitutionor addition into the MTSP polypeptide sequence. Non-classical aminoacids include but are not limited to the D-isomers of the common aminoacids, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyricacid, ∈-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid,3-amino propionoic acid, ornithine, norleucine, norvaline,hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine,fluoro-amino acids, designer amino acids such as β-methyl amino acids,Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs ingeneral. Furthermore, the amino acid can be D (dextrorotary) or L(levorotary).

In cases where natural products are suspected of being mutant or areisolated from new species, the amino acid sequence of the MTSPpolypeptide isolated from the natural source, as well as those expressedin vitro, or from synthesized expression vectors in vivo or in vitro,can be determined from analysis of the DNA sequence, or alternatively,by direct sequencing of the isolated protein. Such analysis can beperformed by manual sequencing or through use of an automated amino acidsequenator.

Modifications

A variety of modifications of the MTSP polypeptides and domains arecontemplated herein. An MTSP-encoding nucleic acid molecule can bemodified by any of numerous strategies known in the art (Sambrook et al.(1990), Molecular Cloning, A Laboratory Manual, 2d ed., Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.). The sequences can becleaved at appropriate sites with restriction endonuclease(s), followedby further enzymatic modification if desired, isolated, and ligated invitro. In the production of the gene encoding a domain, derivative oranalog of MTSP, care should be taken to ensure that the modified generetains the original translational reading frame, uninterrupted bytranslational stop signals, in the gene region where the desiredactivity is encoded.

Additionally, the MTSP-encoding nucleic acid molecules can be mutated invitro or in vivo, to create and/or destroy translation, initiation,and/or termination sequences, or to create variations in coding regionsand/or form new restriction endonuclease sites or destroy pre-existingones, to facilitate further in vitro modification. Also, as describedherein muteins with primary sequence alterations, such as replacementsof Cys residues and elimination or addition of glycosylation sites arecontemplated; the MTSP10 that includes the sequence of amino acids setforth in SEQ ID No. 6 has potential glycosylation sites at N₆₇, N₂₇₂;N₃₃₆; N₃₈₃, N₄₀₉ and N₄₁₈ (SEQ ID No. 23). Mutations can be effected byany technique for mutagenesis known in the art, including, but notlimited to, chemical mutagenesis and in vitro site-directed mutagenesis(Hutchinson et al., J. Biol. Chem. 253:6551–6558 (1978)), use of TAB®linkers (Pharmacia). In one embodiment, for example, an MTSP polypeptideor domain thereof is modified to include a fluorescent label. In otherspecific embodiments, the MTSP polypeptide is modified such thatheterobifunctional reagents can be used to crosslink the members of acomplex.

In addition, domains, analogs and derivatives of an MTSP can bechemically synthesized. For example, a peptide corresponding to aportion of an MTSP, which includes the desired domain or which mediatesthe desired activity in vitro can be synthesized by use of a peptidesynthesizer. Furthermore, if desired, nonclassical amino acids orchemical amino acid analogs can be introduced as a substitution oraddition into the MTSP sequence. Non-classical amino acids include butare not limited to the D-isomers of the common amino acids, a-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, ∈-Abu,e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-aminopropionoic acid, ornithine, norleucine, norvaline, hydroxyproline,sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine,phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids,designer amino acids such as β-methyl amino acids, Ca-methyl aminoacids, Na-methyl amino acids, and amino acid analogs in general.Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

F. Screening Methods

The single chain protease domains, as shown herein, can be used in avariety of methods to identify compounds that modulate the activitythereof. For SPs that exhibit higher activity or expression in tumorcells, compounds that inhibit the proteolytic activity are of particularinterest. For any SPs that are active at lower levels in tumor cells,compounds or agents that enhance the activity are potentially ofinterest. In all instances the identified compounds include agents thatare candidate cancer treatments.

Several types of assays are exemplified and described herein. It isunderstood that the protease domains can be used in other assays. It isshown here, however, that the single chain protease domains exhibitcatalytic activity. As such they are ideal for in vitro screeningassays. They can also be used in binding assays.

The MTSP10 full length zymogens, activated enzymes, single and two chainprotease domains are contemplated for use in any screening assay knownto those of skill in the art, including those provided herein. Hence thefollowing description, if directed to proteolytic assays is intended toapply to use of a single chain protease domain or a catalytically activeportion thereof of any SP, including an MTSP10. Other assays, such asbinding assays are provided herein, particularly for use with an MTSP10,including any variants, such as splice variants thereof.

1. Catalytic Assays for Identification of Agents that Modulate theProtease Activity of an SP Protein

Methods for identifying a modulator of the catalytic activity of an SP,particularly a single chain protease domain or catalytically activeportion thereof, are provided herein. The methods can be practiced by:contacting the MTSP10, a full-length zymogen or activated form, andparticularly a single-chain domain thereof, with a substrate of theMTSP10 in the presence of a test substance, and detecting theproteolysis of the substrate, whereby the activity of the MTSP10 isassessed, and comparing the activity to a control. For example, acontrol can be the activity of the MTSP10 assessed by contacting anMTSP10, including a full-length zymogen or activated form, andparticularly a single-chain domain thereof, particularly a single-chaindomain thereof, with a substrate of the MTSP10, and detecting theproteolysis of the substrate, whereby the activity of the MTSP10 isassessed. The results in the presence and absence of the test compoundsare compared. A difference in the activity indicates that the testsubstance modulates the activity of the MTSP10. Activators of MTSP10activation cleavage are also contemplated; such assays are discussedbelow.

In one embodiment a plurality of the test substances are screenedsimultaneously in the above screening method. In another embodiment, theMTSP10 is isolated from a target cell as a means for then identifyingagents that are potentially specific for the target cell.

In another embodiment, a test substance is a therapeutic compound, andwhereby a difference of the MTSP10 activity measured in the presence andin the absence of the test substance indicates that the target cellresponds to the therapeutic compound.

One method includes the steps of (a) contacting the MTSP10 polypeptideor protease domain thereof with one or a plurality of test compoundsunder conditions conducive to interaction between the ligand and thecompounds; and (b) identifying one or more compounds in the pluralitythat specifically binds to the ligand.

Another method provided herein includes the steps of a) contacting anMTSP10 polypeptide or protease domain thereof with a substrate of theMTSP10 polypeptide, and detecting the proteolysis of the substrate,whereby the activity of the MTSP10 polypeptide is assessed; b)contacting the MTSP10 polypeptide with a substrate of the MTSP10polypeptide in the presence of a test substance, and detecting theproteolysis of the substrate, whereby the activity of the MTSP10polypeptide is assessed; and c) comparing the activity of the MTSP10polypeptide assessed in steps a) and b), whereby the activity measuredin step a) differs from the activity measured in step b) indicates thatthe test substance modulates the activity of the MTSP10 polypeptide.

In another embodiment, a plurality of the test substances are screenedsimultaneously. In comparing the activity of an MTSP10 polypeptide inthe presence and absence of a test substance to assess whether the testsubstance is a modulator of the MTSP10 polypeptide, it is unnecessary toassay the activity in parallel, although such parallel measurement istypical. It is possible to measure the activity of the MTSP10polypeptide at one time point and compare the measured activity to ahistorical value of the activity of the MTSP10 polypeptide.

For instance, one can measure the activity of the MTSP10 polypeptide inthe presence of a test substance and compare with historical value ofthe activity of the MTSP10 polypeptide measured previously in theabsence of the test substance, and vice versa. This can be accomplished,for example, by providing the activity of the MTSP10 polypeptide on aninsert or pamphlet provided with a kit for conducting the assay.

Methods for selecting substrates for a particular SP are described inthe EXAMPLES, and particular proteolytic assays are exemplified.

Combinations and kits containing the combinations optionally includinginstructions for performing the assays are provided. The combinationsinclude an MTSP10 polypeptide and a substrate of the MTSP10 polypeptideto be assayed; and, optionally reagents for detecting proteolysis of thesubstrate. The substrates, which can be chromogenic or fluorgenicmolecules, including proteins, subject to proteolysis by a particularMTSP10 polypeptide, can be identified empirically by testing the abilityof the MTSP10 polypeptide to cleave the test substrate. Substrates thatare cleaved most effectively (i.e., at the lowest concentrations and/orfastest rate or under desirable conditions), are identified.

Additionally provided herein is a kit containing the above-describedcombination. The kit optionally includes instructions for identifying amodulator of the activity of an MTSP10 polypeptide. Any MTSP10polypeptide is contemplated as target for identifying modulators of theactivity thereof.

2. Binding Assays

Also provided herein are methods for identification and isolation ofagents, particularly compounds that bind to MTSP10s. The assays aredesigned to identify agents that bind to the zymogen form, the singlechain isolated protease domain (or a protein, other than an MTSP10polypeptide, that contains the protease domain of an MTSP10polypeptide), and to the activated form, including the activated formderived from the full length zymogen or from an extended proteasedomain. The identified compounds are candidates or leads foridentification of compounds for treatments of tumors and other disordersand diseases involving aberrant angiogenesis. The MTSP10 polypeptidesused in the methods include any MTSP10 polypeptide as defined herein,including the MTSP10 single chain protease domain or proteolyticallyactive portion thereof.

A variety of methods are provided herein. These methods can be performedin solution or in solid phase reactions in which the MTSP10polypeptide(s) or protease domain(s) thereof are linked, either directlyor indirectly via a linker, to a solid support. Screening assays aredescribed in the Examples, and these assays have been used to identifycandidate compounds. For purposes herein, all binding assays describedabove are provided for MTSP10.

Methods for identifying an agent, such as a compound, that specificallybinds to an MTSP10 single and/or two chain protease domain, a zymogenand/or full-length activated MTSP10 or two chain protease domain thereofare provided herein. The method can be practiced by

-   (a) contacting the MTSP10 with one or a plurality of test agents    under conditions conducive to binding between the MTSP10 and an    agent; and-   (b) identifying one or more agents within the plurality that    specifically binds to the MTSP10.

For example, in practicing such methods the MTSP10 polypeptide is mixedwith a potential binding partner or an extract or fraction of a cellunder conditions that allow the association of potential bindingpartners with the polypeptide. After mixing, peptides, polypeptides,proteins or other molecules that have become associated with an MTSP10are separated from the mixture. The binding partner that bound to theMTSP10 can then be removed and further analyzed. To identify and isolatea binding partner, the entire protein, for instance the entirepolypeptided of SEQ ID Nos. 6 can be used. Alternatively, a fragment ofthe protein can be used.

A variety of methods can be used to obtain cell extracts or body fluids,such as blood, serum, urine, sweat, synovial fluid, CSF and other suchfluids. For example, cells can be disrupted using either physical orchemical disruption methods. Examples of physical disruption methodsinclude, but are not limited to, sonication and mechanical shearing.Examples of chemical lysis methods include, but are not limited to,detergent lysis and enzyme lysis. A skilled artisan can readily adaptmethods for preparing cellular extracts in order to obtain extracts foruse in the present methods.

Once an extract of a cell is prepared, the extract is mixed with theMTSP10 under conditions in which association of the protein with thebinding partner can occur. A variety of conditions can be used,including conditions that resemble conditions found in the cytoplasm ofa human cell or in a body fluid, such as blood. Features, such asosmolarity, pH, temperature, and the concentration of cellular extractused, can be varied to optimize the association of the protein with thebinding partner. Similarly, methods for isolation of molecules ofinterest from body fluids are known.

After mixing under appropriate conditions, the bound complex isseparated from the mixture. A variety of techniques can be used toseparate the mixture. For example, antibodies specific to an MTSP10 canbe used to immunoprecipitate the binding partner complex. Alternatively,standard chemical separation techniques such as chromatography anddensity/sediment centrifugation can be used.

After removing the non-associated cellular constituents in the extract,the binding partner can be dissociated from the complex usingconventional methods. For example, dissociation can be accomplished byaltering the salt concentration or pH of the mixture.

To aid in separating associated binding partner pairs from the mixedextract, the MTSP10 can be immobilized on a solid support. For example,the protein can be attached to a nitrocellulose matrix or acrylic beads.Attachment of the protein or a fragment thereof to a solid support aidsin separating peptide/binding partner pairs from other constituentsfound in the extract. The identified binding partners can be either asingle protein or a complex made up of two or more proteins.

Alternatively, the nucleic acid molecules encoding the single chainproteases can be used in a yeast two-hybrid system. The yeast two-hybridsystem has been used to identify other protein partner pairs and canreadily be adapted to employ the nucleic acid molecules hereindescribed.

Another in vitro binding assay, particularly for an MTSP10, uses amixture of a polypeptide that contains at least the catalytic domain ofone of these proteins and one or more candidate binding targets orsubstrates. After incubating the mixture under appropriate conditions,the ability of the MTSP10 or a polypeptide fragment thereof containingthe catalytic domain to bind to or interact with the candidate substrateis assessed. For cell-free binding assays, one of the componentsincludes or is coupled to a detectable label. The label can provide fordirect detection, such as radioactivity, luminescence, optical orelectron density, etc., or indirect detection such as an epitope tag, anenzyme, etc. A variety of methods can be employed to detect the labeldepending on the nature of the label and other assay components. Forexample, the label can be detected bound to the solid substrate or aportion of the bound complex containing the label can be separated fromthe solid substrate, and the label thereafter detected.

3. Detection of Signal Transduction

MTSP10, which is a transmembrane protein, can be involved directly orindirectly in signal transduction directly as a cell surface receptor orindirectly by activating proteins, such as pro-growth factors that caninitiate signal transduction.

In addition, secretion of MTSP10, such as the extracellular domain ofMTSP10, can be involved in signal transduction either directly bybinding to or interacting with a cell surface receptor or indirectly byactivating proteins, such as pro-growth factors that can initiate signaltransduction. Assays for assessing signal transduction are well known tothose of skill in the art, and can be adapted for use with the MTSP10polypeptide.

Assays for identifying agents that affect or alter signal transductionmediated directly or indirectly, such as via activation of a pro-growthfactor, by an MTSP10, particularly the full length or a sufficientportion to anchor the extracellular domain or a functional portionthereof of an MTSP10 on the surface of a cell are provided. Such assays,include, for example, transcription based assays in which modulation ofa transduced signal is assessed by detecting an effect on an expressionfrom a reporter gene (see, e.g., U.S. Pat. No. 5,436,128).

4. Methods for Identifying Agents that Modulate the Expression a NucleicAcid Encoding an MTSP10

Another embodiment provides methods for identifying agents that modulatethe expression of a nucleic acid encoding an MTSP10. Such assays use anyavailable means of monitoring for changes in the expression level of thenucleic acids encoding an MTSP10.

In one assay format, cell lines that contain reporter gene fusionsbetween the open reading frame of MTSP10 or a domain thereof,particularly the protease domain and any assayable fusion partner can beprepared. Numerous assayable fusion partners are known and readilyavailable including the firefly luciferase gene and the gene encodingchloramphenicol acetyltransferase (Alam et al., Anal. Biochem.188:245–54 (1990)). Cell lines containing the reporter gene fusions arethen exposed to the agent to be tested under appropriate conditions andtime. Differential expression of the reporter gene between samplesexposed to the agent and control samples identifies agents whichmodulate the expression of a nucleic acid encoding an MTSP10.

Additional assay formats can be used to monitor the ability of the agentto modulate the expression of a nucleic acid encoding an MTSP10. Forinstance, mRNA expression can be monitored directly by hybridization tothe nucleic acids. Cell lines are exposed to the agent to be testedunder appropriate conditions and time and total RNA or mRNA is isolatedby standard procedures (see, e.g., Sambrook et al. (1989) MOLECULARCLONING: A LABORATORY MANUAL, 2nd Ed. Cold Spring Harbor LaboratoryPress). Probes to detect differences in RNA expression levels betweencells exposed to the agent and control cells can be prepared from thenucleic acids. It is typical, but not necessary, to design probes whichhybridize only with target nucleic acids under conditions of highstringency. Only highly complementary nucleic acid hybrids form underconditions of high stringency. Accordingly, the stringency of the assayconditions determines the amount of complementarity which should existbetween two nucleic acid strands in order to form a hybrid. Stringencyshould be chosen to maximize the difference in stability between theprobe:target hybrid and potential probe:non-target hybrids.

Probes can be designed from the nucleic acids through methods known inthe art. For instance, the G+C content of the probe and the probe lengthcan affect probe binding to its target sequence. Methods to optimizeprobe specificity are commonly available (see, e.g., Sambrook et al.(1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed. Cold SpringHarbor Laboratory Press); and Ausubel et al. (1995) CURRENT PROTOCOLS INMOLECULAR BIOLOGY, Greene Publishing Co., NY).

Hybridization conditions are modified using known methods (see, e.g.,Sambrook et al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.Cold Spring Harbor Laboratory Press); and Ausubel et al. (1995) CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Co., NY), as requiredfor each probe. Hybridization of total cellular RNA or RNA enriched forpolyA RNA can be accomplished in any available format. For instance,total cellular RNA or RNA enriched for polyA RNA can be affixed to asolid support, and the solid support exposed to at least one probecomprising at least one, or part of one of the nucleic acid moleculesunder conditions in which the probe specifically hybridizes.Alternatively, nucleic acid fragments comprising at least one, or partof one of the sequences can be affixed to a solid support, such as aporous glass wafer. The glass wafer can then be exposed to totalcellular RNA or polyA RNA from a sample under conditions in which theaffixed sequences specifically hybridize. Such glass wafers andhybridization methods are widely available, for example, those disclosedby Beattie (WO 95/11755). By examining for the ability of a given probeto specifically hybridize to an RNA sample from an untreated cellpopulation and from a cell population exposed to the agent, agents whichup or down regulate the expression of a nucleic acid encoding the MTSP10polypeptide, are identified.

In one format, the relative amounts of a protein between a cellpopulation that has been exposed to the agent to be tested compared toan un-exposed control cell population can be assayed (e.g., a prostatecancer cell line, a lung cancer cell line, a colon cancer cell line or abreast cancer cell line). In this format, probes, such as specificantibodies, are used to monitor the differential expression or level ofactivity of the protein in the different cell populations or bodyfluids. Cell lines or populations or body fluids are exposed to theagent to be tested under appropriate conditions and time. Cellularlysates or body fluids can be prepared from the exposed cell line orpopulation and a control, unexposed cell line or population or unexposedbody fluid. The cellular lysates or body fluids are then analyzed withthe probe.

For example, N- and C-terminal fragments of the MTSP10 can be expressedin bacteria and used to search for proteins which bind to thesefragments. Fusion proteins, such as His-tag or GST fusion to the N- orC-terminal regions of the MTSP10 can be prepared for use as a substrate.These fusion proteins can be coupled to, for example,Glutathione-Sepharose beads and then probed with cell lysates or bodyfluids. Prior to lysis, the cells or body fluids can be treated with acandidate agent which can modulate an MTSP10 or proteins that interactwith domains thereon. Lysate proteins binding to the fusion proteins canbe resolved by SDS-PAGE, isolated and identified by protein sequencingor mass spectroscopy, as is known in the art.

Antibody probes are prepared by immunizing suitable mammalian hosts inappropriate immunization protocols using the peptides, polypeptides orproteins if they are of sufficient length (e.g., 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 20, 25, 30, 35, 40 or more consecutive amino acidsthe MTSP10 polypeptide or if required to enhance immunogenicity,conjugated to suitable carriers. Methods for preparing immunogenicconjugates with carriers, such as bovine serum albumin (BSA), keyholelimpet hemocyanin (KLH), or other carrier proteins are well known in theart. In some circumstances, direct conjugation using, for example,carbodiimide reagents can be effective; in other instances linkingreagents such as those supplied by Pierce Chemical Co., Rockford, Ill.,can be desirable to provide accessibility to the hapten. Hapten peptidescan be extended at either the amino or carboxy terminus with a Cysresidue or interspersed with cysteine residues, for example, tofacilitate linking to a carrier. Administration of the immunogens isconducted generally by injection over a suitable time period and withuse of suitable adjuvants, as is generally understood in the art. Duringthe immunization schedule, titers of antibodies are taken to determineadequacy of antibody formation.

Anti-peptide antibodies can be generated using synthetic peptidescorresponding to, for example, the carboxy terminal amino acids of theMTSP10. Synthetic peptides can be as small as 1–3 amino acids in length,generally at least 4 or more amino acid residues long. The peptides canbe coupled to KLH using standard methods and can be immunized intoanimals, such as rabbits or ungulates. Polyclonal antibodies can then bepurified, for example using Actigel beads containing the covalentlybound peptide.

While the polyclonal antisera produced in this way can be satisfactoryfor some applications, for pharmaceutical compositions, use ofmonoclonal preparations are generally used. Immortalized cell lineswhich secrete the desired monoclonal antibodies can be prepared usingthe standard method of Kohler et al., (Nature 256:495–7 (1975)) ormodifications which effect immortalization of lymphocytes or spleencells, as is generally known. The immortalized cell lines secreting thedesired antibodies are screened by immunoassay in which the antigen isthe peptide hapten, polypeptide or protein. When the appropriateimmortalized cell culture secreting the desired antibody is identified,the cells can be cultured either in vitro or by production in vivo viaascites fluid. Of particular interest, are monoclonal antibodies thatrecognize the catalytic domain or activation cleavage site (region) ofan MTSP10.

Additionally, the zymogen or two-chain form of the MTSP10 can be used tomake monoclonal antibodies that recognize conformation epitopes. Thedesired monoclonal antibodies are then recovered from the culturesupernatant or from the ascites supernatant. Fragments of themonoclonals or the polyclonal antisera which contain the immunologicallysignificant portion can be used as antagonists, as well as the intactantibodies. Use of immunologically reactive fragments, such as the Fab,Fab′, of F(ab′)₂ fragments are often used, especially in a therapeuticcontext, as these fragments are generally less immunogenic than thewhole immunoglobulin.

The antibodies or fragments can also be produced. Regions that bindspecifically to the desired regions of receptor also can be produced inthe context of chimeras with multiple species origin.

Agents that are assayed in the above method can be randomly selected orrationally selected or designed.

The agents can be, as examples, peptides, small molecules, andcarbohydrates. A skilled artisan can readily recognize that there is nolimit as to the structural nature of the agents.

The peptide agents can be prepared using standard solid phase (orsolution phase) peptide synthesis methods, as is known in the art. Inaddition, the DNA encoding these peptides can be synthesized usingcommercially available oligonucleotide synthesis instrumentation andproduced recombinantly using standard recombinant production systems.The production using solid phase peptide synthesis is necessitated ifnon-gene-encoded amino acids are to be included.

G. Assay Formats and Selection of Test Substances that Modulate at LeastOne Activity of an MTSP10 Polypeptide

Methods for identifying agents that modulate at least one activity of anMTSP10 are provided. The methods include phage display and other methodsfor assessing alterations in the activity of an MTSP10. Such methods orassays can use any means of monitoring or detecting the desiredactivity. A variety of formats and detection protocols are known forperforming screening assays. Any such formats and protocols can beadapted for identifying modulators of MTSP10 polypeptide activities. Thefollowing includes a discussion of exemplary protocols.

1. High Throughput Screening Assays

Although the above-described assay can be conducted where a singleMTSP10 polypeptide is screened, and/or a single test substance isscreened in one assay, the assay typically is conducted in a highthroughput screening mode, i.e., a plurality of the SP proteins arescreened against and/or a plurality of the test substances are screenedsimultaneously (See generally, High Throughput Screening: The Discoveryof Bioactive Substances (Devlin, Ed.) Marcel Dekker, 1997; Sittampalamet al., Curr. Opin. Chem. Biol., 1:384–91 (1997); and Silverman et al.,Curr. Opin. Chem. Biol., 2:397–403 (1998)). For example, the assay canbe conducted in a multi-well (e.g., 24-, 48-, 96-, 384-, 1536-well orhigher density), chip or array format.

High-throughput screening (HTS) is the process of testing a large numberof diverse chemical structures against disease targets to identify“hits” (Sittampalam et al., Curr. Opin. Chem. Biol., 1:384–91 (1997)).Current state-of-the-art HTS operations are highly automated andcomputerized to handle sample preparation, assay procedures and thesubsequent processing of large volumes of data.

Detection technologies employed in high-throughput screens depend on thetype of biochemical pathway being investigated (Sittampalam et al.,Curr. Opin. Chem. Biol., 1:384–91 (1997)). These methods include,radiochemical methods, such as the scintillation proximity assays (SPA),which can be adapted to a variety of enzyme assays (Lerner et al., J.Biomol. Screening, 1:135–143 (1996); Baker et al., Anal. Biochem.,239:20–24 (1996); Baum et al., Anal. Biochem., 237:129–134 (1996); andSullivan et al., J. Biomol. Screening 2:19–23 (1997)) andprotein-protein interaction assays (Braunwalder et al., J. Biomol.Screening 1:23–26 (1996); Sonatore et al., Anal. Biochem. 240:289–297(1996); and Chen et al., J. Biol. Chem. 271:25308–25315 (1996)), andnon-isotopic detection methods, including but are not limited to,colorimetric and luminescence detection methods, resonance energytransfer (RET) methods, time-resolved fluorescence (HTRF) methods,cell-based fluorescence assays, such as fluorescence resonance energytransfer (FRET) procedures (see, e.g., Gonzalez et al., Biophys. J.,69:1272–1280 (1995)), fluorescence polarization or anisotropy methods(see, e.g., Jameson et al., Methods Enzymol. 246:283–300 (1995); Jolley,J. Biomol. Screening 1:33–38 (1996); Lynch et al., Anal. Biochem.247:77–82 (1997)), fluorescence correlation spectroscopy (FCS) and othersuch methods.

2. Test Substances

Test compounds, including small molecules, antibodies, proteins, nucleicacids, peptides, natural products, extracts containing natural productsand libraries and collections thereof, can be screened in theabove-described assays and assays described below to identify compoundsthat modulate the activity of an MTSP10 polypeptide. Rational drugdesign methodologies that rely on computational chemistry can be used toscreen and identify candidate compounds.

The compounds identified by the screening methods include inhibitors,including antagonists, and can be agonists. Compounds for screeninginclude any compounds and collections of compounds available, known orthat can be prepared.

a. Selection of Compounds

Compounds can be selected for their potency and selectivity ofinhibition of serine proteases, especially an MTSP10 polypeptide. Asdescribed herein, and as generally known, a target serine protease andits substrate are combined under assay conditions permitting reaction ofthe protease with its substrate. The assay is performed in the absenceof test compound, and in the presence of increasing concentrations ofthe test compound. The concentration of test compound at which 50% ofthe serine protease activity is inhibited by the test compound is theIC₅₀ value (Inhibitory Concentration) or EC₅₀ (Effective Concentration)value for that compound. Within a series or group of test compounds,those having lower IC₅₀ or EC₅₀ values are considered more potentinhibitors of the serine protease than those compounds having higherIC₅₀ or EC₅₀ values. The IC₅₀ measurement is often used for moresimplistic assays,. whereas the EC₅₀ is often used for more complicatedassays, such as those employing cells.

Typically candidate compounds have an IC₅₀ value of 100 nM or less asmeasured in an in vitro assay for inhibition of MTSP10 polypeptideactivity. The test compounds also are evaluated for selectivity toward aserine protease. As described herein, and as generally known, a testcompound is assayed for its potency toward a panel of serine proteasesand other enzymes and an IC₅₀ value or EC₅₀ value is determined for eachtest compound in each assay system. A compound that demonstrates a lowIC₅₀ value or EC₅₀ value for the target enzyme, e.g., MTSP10polypeptide, and a higher IC₅₀ value or EC₅₀ value for other enzymeswithin the test panel (e.g., urokinase tissue plasminogen activator,thrombin, Factor Xa), is considered to be selective toward the targetenzyme. Generally, a compound is deemed selective if its IC₅₀ value orEC₅₀ value in the target enzyme assay is at least one order of magnitudeless than the next smallest IC₅₀ value or EC₅₀ value measured in theselectivity panel of enzymes.

Compounds are also evaluated for their activity in vivo. The type ofassay chosen for evaluation of test compounds depends on thepathological condition to be treated or prevented by use of thecompound, as well as the route of administration to be evaluated for thetest compound.

For instance, to evaluate the activity of a compound to reduce tumorgrowth through inhibition of MTSP10 polypeptide, the proceduresdescribed by Jankun et al., Canc. Res. 57:559–563 (1997) to evaluatePAI-1 can be employed. Briefly, the ATCC cell lines DU145 and LnCaP areinjected into SCID mice. After tumors are established, the mice aregiven test compound according to a dosing regime determined from thecompound's in vitro characteristics. The Jankun et al. compound wasadministered in water. Tumor volume measurements are taken twice a weekfor about five weeks. A compound is deemed active if an animal to whichthe compound was administered exhibited decreased tumor volume, ascompared to animals receiving appropriate control compounds.

Another in vivo experimental model designed to evaluate the effect ofp-aminobenzamidine, a swine protease inhibitor, on reducing tumor volumeis described by Billström et al., Int. J. Cancer 61:542–547 (1995).

To evaluate the ability of a compound to reduce the occurrence of, orinhibit, metastasis, the procedures described by Kobayashi et al. Int.J. Canc. 57:727–733d (1994) can be employed. Briefly, a murine xenograftselected for high lung colonization potential in injected into C57B1/6mice i.v. (experimental metastasis) or s.c. into the abdominal wall(spontaneous metastasis). Various concentrations of the compound to betested can be admixed with the tumor cells in Matrigel prior toinjection. Daily i.p. injections of the test compound are made either ondays 1–6 or days 7–13 after tumor inoculation. The animals aresacrificed about three or four weeks after tumor inoculation, and thelung tumor colonies are counted. Evaluation of the resulting datapermits a determination as to efficacy of the test compound, optimaldosing and route of administration.

The activity of the tested compounds toward decreasing tumor volume andmetastasis can be evaluated in model described in Rabbani et al., Int.J. Cancer 63:840–845 (1995) to evaluate their inhibitor. There, Mat LyLutumor cells were injected into the flank of Copenhagen rats. The animalswere implanted with osmotic minipumps to continuously administer variousdoses of test compound for up to three weeks. The tumor mass and volumeof experimental and control animals were evaluated during theexperiment, as were metastatic growths. Evaluation of the resulting datapermits a determination as to efficacy of the test compound, optimaldosing, and route of administration. Some of these authors described arelated protocol in Xing et al., Canc. Res. 57:3585–3593 (1997).

To evaluate the anti-angiogenesis activity of a compound, a rabbitcornea neovascularization model can be employed (see, e.g., Avery et al.(1990) Arch. Ophthalmol., 108:1474–147). Avery et al. describesanesthetizing New Zealand albino rabbits and then making a centralcorneal incision and forming a radial corneal pocket. A slow releaseprostaglandin pellet was placed in the pocket to induceneovascularization. Test compound was administered i.p. for five days,at which time the animals were sacrificed. The effect of the testcompound is evaluated by review of periodic photographs taken of thelimbus, which can be used to calculate the area of neovascular responseand, therefore, limbal neovascularization. A decreased area ofneovascularization as compared with appropriate controls indicates thetest compound was effective at decreasing or inhibitingneovascularization.

An angiogenesis model used to evaluate the effect of a test compound inpreventing angiogenesis is described by Min et al. Canc. Res.56:2428–2433 (1996). C57BL6 mice receive subcutaneous injections of aMatrigel mixture containing bFGF, as the angiogenesis-inducing agent,with and without the test compound. After five days, the animals aresacrificed and the Matrigel plugs, in which neovascularization can bevisualized, are photographed. An experimental animal receiving Matrigeland an effective dose of test compound exhibits less vascularizationthan a control animal or an experimental animal receiving a less- ornon-effective does of compound.

An in vivo system designed to test compounds for their ability to limitthe spread of primary tumors is described by Crowley et al., Proc. Natl.Acad. Sci. 90:5021–5025 (1993). Nude mice are injected with tumor cells(PC3) engineered to express CAT (chloramphenicol acetyltransferase).Compounds to be tested for their ability to decrease tumor size and/ormetastases are administered to the animals, and subsequent measurementsof tumor size and/or metastatic growths are made. In addition, the levelof CAT detected in various organs provides an indication of the abilityof the test compound to inhibit metastasis; detection of less CAT intissues of a treated animal versus a control animal indicates lessCAT-expressing cells migrated to that tissue.

In vivo experimental models designed to evaluate the inhibitorypotential of a test serine protease inhibitors, using a tumor cell lineF3II known to be highly invasive (see, e.g., Alonso et al. (1996) BreastCanc. Res. Treat. 40:209–223) are provided. Alonso describes in vivostudies for toxicity determination, tumor growth, invasiveness,spontaneous metastasis, experimental lung metastasis, and anangiogenesis assay.

The CAM model (chick embryo chorioallantoic membrane model; Ossowski(1988) J. Cell Biol. 107:2437–2445), provides another method forevaluating the inhibitory activity of a test compound. In the CAM model,tumor cells invade through the chorioallantoic membrane containing CAM(with tumor cells in the presence of several serine protease inhibitorsresults in less or no invasion of the tumor cells through the membrane).Thus, the CAM assay is performed with CAM and tumor cells in thepresence and absence of various concentrations of test compound. Theinvasiveness of tumor cells is measured under such conditions to providean indication of the compound's inhibitory activity. A compound havinginhibitory activity correlates with less tumor invasion.

The CAM model is also used in a standard assay of angiogenesis (i.e.,effect on formation of new blood vessels (Brooks et al. Methods inMolecular Biology 129:257–269 (1999)). According to this model, a filterdisc containing an angiogenesis inducer, such as basic fibroblast growthfactor (bFGF) is placed onto the CAM. Diffusion of the cytokine into theCAM induces local angiogenesis, which can be measured in several wayssuch as by counting the number of blood vessel branch points within theCAM directly below the filter disc. The ability of identified compoundsto inhibit cytokine-induced angiogenesis can be tested using this model.A test compound can either be added to the filter disc that contains theangiogenesis inducer, be placed directly on the membrane or beadministered systemically. The extent of new blood vessel formation inthe presence and/or absence of test compound can be compared using thismodel. The formation of fewer new blood vessels in the presence of atest compound would be indicative of anti-angiogenesis activity.Demonstration of anti-angiogenesis activity for inhibitors of an MTSP10polypeptide indicates a role in angiogenesis for that SP protein.

b. Known Serine Protease Inhibitors

Compounds for screening can be serine protease inhibitors, which can betested for their ability to inhibit the activity of an MTSP10.Exemplary, serine protease inhibitors for use in the screening assays,include, but are not limited to: Serine Protease Inhibitor 3 (SPI-3)(Chen, et al. Citokine, 11:856–862 (1999)); Aprotinin (lijima, R., etal., J. Biochem. (Tokyo) 126:912–916 (1999)); Kazal-type serine proteaseinhibitor-like proteins (Niimi, et al. Eur. J. Biochem., 266:282–292(1999)); Kunitz-type serine protease inhibitor (Ravichandran, S., etal., Acta Crystallogr. D. Biol. Crystallogr., 55:1814–1821 (1999));Tissue factor pathway inhibitor-2/Matrix-associated serine roteaseinhibitor (TFPI-2/MSPI), (Liu, Y. et al. Arch. Biochem. Biophys.370:112–8 (1999)); Bukunin (Cui, C. Y. et al. J. Invest. Dermatol.113:182–8 (1999)); Nafmostat mesilate (Ryo, R. et al. Vox Sang. 76:241–6(1999)); TPCK (Huang et al. Oncogene 18:3431–3439 (1999)); A syntheticcotton-bound serine protease inhibitor (Edwards (1999) et al WoundRepair Regen. 7:106–18); FUT-175 (Sawada (1999) et al. Stroke30:644–50); Combination of serine protease inhibitor FUT-0175 andthromboxane synthetase inhibitor OKY-046 (Kaminogo et al. (1998) Neurol.Med. Chir. (Tokyo) 38:704–8; discussion 708–9); the rat serine proteaseinhibitor 2.1 gene (LeCam, A., et al., Biochem. Biophys. Res. Commun.,253:311–4 (1998)); A new intracellular serine protease inhibitorexpressed in the rat pituitary gland complexes with granzyme B (Hill etal. FEBS Lett. 440:361–4 (1998)); 3,4-Dichloroisocoumarin (Hammed et al.Proc. Soc. Exp. Biol. Med., 219:132–7 (1998)); LEX032 (Bains et al. Eur.J. Pharmacol. 356:67–72 (1998)); N-tosyl-L-phenylalanine chloromethylketone (Dryjanski et al. Biochemistry 37:14151–6 (1998)); Mouse gene forthe serine protease inhibitor neuroserpin (P112) (Berger et al. Gene,214:25–33 (1998)); Rat serine protease inhibitor 2.3 gene (Paul et al.Eur. J. Biochem. 254:538–46 (1998)); Ecotin (Yang et al. J. Mol. Biol.279:945–57 (1998)); A 14 kDa plant-related serine protease inhibitor(Roch et al. Dev. Comp. Immunol. 22(1):1–12 (1998)); Matrix-associatedserine protease inhibitor TFPI-2/33 kDa MSPI (Rao et al. Int. J. Cancer76:749–56 (1998)); ONO-3403 (Hiwasa et al. Cancer Lett. 126:221–5(1998)); Bdellastasin (Moser et al. Eur. J. Biochem. 253:212–20 (1998));Bikunin (Xu et al. J. Mol. Biol. 276:955–66 (1998)); Nafamostat mesilate(Meligren et al. Thromb. Haemost. 79:342–7 (1998)); The growth hormonedependent serine protease inhibitor, Spi 2.1 (Maake et al. Endocrinology138:5630–6 (1997)); Growth factor activator inhibitor type 2, aKunitz-type serine protease inhibitor (Kawaguchi et al. J. Biol. Chem.,272:27558–64 (1997)); Heat-stable serine protease inhibitor protein fromovaries of the desert locust, Schistocerga gregaria (Hamdaoui et al.Biochem. Biophys. Res. Commun. 238:357–60 (1997)); Human placentalHepatocyte growth factor activator inhibitor, a Kunitz-type serineprotease inhibitor (Shimomura et al. J. Biol. Chem. 272:6370–6 (1997));FUT-187, oral serine protease inhibitor (Shiozaki et al. Gan To KagukuRyoho, 23(14): 1971–9 (1996)); Extracellular matrix-associated serineprotease inhibitors (Mr 33,000, 31,000, and 27,000 (Rao, C. N., et al.,Arch. Biochem. Biophys., 335:82–92 (1996)); An irreversible isocoumarinserine protease inhibitor (Palencia, D. D., et al., Biol. Reprod.,55:536–42 (1996)); 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF)(Nakabo et al. J. Leukoc. Biol. 60:328–36 (1996)); Neuroserpin(Osterwalder, T., et al., EMBO J. 15:2944–53 (1996)); Human serineprotease inhibitor alpha-1-antitrypsin (Forney et al. J. Parasitol.82:496–502 (1996)); Rat serine protease inhibitor 2.3 (Simar-Blanchet,A. E., et al., Eur. J. Biochem., 236:638–48 (1996)); Gebaxate mesilate(parodi, F., et al., J. Cardiothorac. Vasc. Anesth. 10:235–7 (1996));Recombinant serine protease inhibitor, CPTI II (Stankiewicz, M., et al.,(Acta Biochim. Pol., 43(3):525–9 (1996)); A cysteine-rich serineprotease inhibitor (Guamerin 11) (Kim, D. R., et al., J. Enzym. Inhib.,10:81–91 (1996)); Diisopropylfluorophosphate (Lundqvist, H., et al.,Inflamm. Res., 44(12):510–7 (1995)); Nexin 1 (Yu, D. W., et al., J. CellSci., 108(Pt 12):3867–74 (1995)); LEX032 (Scalia, R., et al., Shock,4(4):251–6 (1995)); Protease nexin I (Houenou, L. J., et al., Proc.Natl. Acad. Sci. U.S.A., 92(3):895–9 (1995)); Chymase-directed serineprotease inhibitor (Woodard S. L., et al., J. Immunol., 153(11):5016–25(1994)); N-alpha-tosyl-L-lysyl-chloromethyl ketone (TLCK) (Bourinbaiar,A. S., et al., Cell Immunol., 155(1):230–6 (1994)); Smpi56 (Ghendler,Y., et al., Exp. Parasitol., 78(2):121–31 (1994)); Schistosomahaematobium serine protease (Blanton, R. E., et al., Mol. Biochem.Parasitol., 63(1):1–11 (1994)); Spi-1 (Warren, W. C., et al., Mol. CellEndocrinol., 98(1):27–32 (1993)); TAME (Jessop, J. J., et al.,Inflammation, 17(5):613–31 (1993)); Antithrombin III (Kalaria, R. N., etal., Am. J. Pathol., 143(3):886–93 (1993)); FOY-305 (Ohkoshi, M., etal., Anticancer Res., 13(4):963–6 (1993)); Camostat mesilate (Senda, S.,et al., Intern. Med., 32(4):350–4 (1993)); Pigment epithelium-derivedfactor (Steele, F. R., et al., Proc. Natl. Acad. Sci. U.S.A.,90(4):1526–30 (1993)); Antistasin (Holstein, T. W., et al., FEBS Lett.,309(3):288–92 (1992)); The vaccinia virus K2L gene encodes a serineprotease inhibitor (Zhou, J., et al., Virology, 189(2):678–86 (1992));Bowman-Birk serine-protease inhibitor (Werner, M. H., et al., J. Mol.Biol., 225(3):873–89 (1992); FUT-175 (Yanamoto, H., et al.,Neurosurgery, 30(3):358–63 (1992)); FUT-175; (Yanamoto, H., et al.,Neurosurgery, 30(3):351–6, discussion 356–7 (1992)); PAI-I (Yreadwell,B. V., et al., J. Orthop. Res., 9(3):309–16 (1991));3,4-Dichloroisocoumarin (Rusbridge, N. M., et al., FEBS Lett.,268(1):133–6 (1990)); Alpha 1-antichymotrypsin (Lindmark, B. E., et al.,Am. Rev. Respir. Des., 141(4 Pt 1):884–8 (1990));P-toluenesulfonyl-L-arginine methyl ester (TAME) (Scuderi, P., J.Immunol., 143(1):168–73 (1989)); Alpha 1-antichymotrypsin (Abraham, C.R., et al., Cell, 52(4):487–501 (1988)); Contrapsin (Modha, J., et al.,Parasitology, 96 (Pt 1):99–109 (1988)); Alpha 2-antiplasmin (Holmes, W.E., et al., J. Biol. Chem., 262(4):1659–64 (1987));3,4-dichloroisocoumarin (Harper, J. W., et al., Biochemistry,24(8):1831–41 (1985)); Diisoprophylfluorophosphate (Tsutsui, K., et al.,Biochem. Biophys. Res. Commun., 123(1):271–7 (1984)); Gabexate mesilate(Hesse, B., et al.,Pharmacol. Res. Commun., 16(7):637–45 (1984)); Phenylmethyl sulfonyl fluoride (Dufer, J., et al., Scand. J. Haematol.,32(1):25–32 (1984)); Protease inhibitor CI-2 (McPhalen, C. A., et al.,J. Mol. Biol., 168(2):445–7 (1983)); Phenylmethylsulfonyl fluoride(Sekar V., et al., Biochem. Biophys. Res. Commun., 89(2):474–8 (1979));PGE1 (Feinstein, M. D., et al., Prostaglandine, 14(6):1075–93 (1977).

c. Combinatorial Libraries and Other Libraries

The source of compounds for the screening assays, can be libraries,including, but are not limited to, combinatorial libraries. Methods forsynthesizing combinatorial libraries and characteristics of suchcombinatorial libraries are known in the art (See generally,Combinatorial Libraries: Synthesis, Screening and Application Potential(Cortese Ed.) Walter de Gruyter, Inc., 1995; Tietze and Lieb, Curr.Opin. Chem. Biol., 2(3):363–71 (1998); Lam, Anticancer Drug Des.,12(3):145–67 (1997); Blaney and Martin, Curr. Opin. Chem. Biol.,1(1):54–9 (1997); and Schultz and Schultz, Biotechnol. Prog.,12(6):729–43 (1996)).

Methods and strategies for generating diverse libraries, primarilypeptide- and nucleotide-based oligomer libraries, have been developedusing molecular biology methods and/or simultaneous chemical synthesismethodologies (see, e.g., Dower et al., Annu. Rep. Med. Chem.,26:271–280 (1991); Fodor et al., Science, 251:767–773 (1991); Jung etal., Angew. Chem. Ind. Ed. Engl., 31:367–383 (1992); Zuckerman et al.,Proc. Natl. Acad. Sci. USA, 89:4505–4509 (1992); Scott et al., Science,249:386–390 (1990); Devlin et al., Science, 249:404–406 (1990); Cwirlaet al., Proc. Natl. Acad. Sci. USA, 87:6378–6382 (1990); and Gallop etal., J. Medicinal Chemistry, 37:1233–1251 (1994)). The resultingcombinatorial libraries potentially contain millions of compounds andthat can be screened to identify compounds that exhibit a selectedactivity.

The libraries fall into roughly three categories:fusion-protein-displayed peptide libraries in which random peptides orproteins are presented on the surface of phage particles or proteinsexpressed from plasmids; support-bound synthetic chemical libraries inwhich individual compounds or mixtures of compounds are presented oninsoluble matrices, such as resin beads (see, e.g., Lam et al., Nature,354:82–84 (1991)) and cotton supports (see, e.g., Eichler et al.,Biochemistry 32:11035–11041 (1993)); and methods in which the compoundsare used in solution (see, e.g., Houghten et al., Nature, 354:84–86(1991); Houghten et al., BioTechniques, 313:412–421 (1992); and Scott etal., Curr. Opin. Biotechnol., 5:40–48 (1994)). There are numerousexamples of synthetic peptide and oligonucleotide combinatoriallibraries and there are many methods for producing libraries thatcontain non-peptidic small organic molecules. Such libraries can bebased on a basis set of monomers that are combined to form mixtures ofdiverse organic molecules or that can be combined to form a librarybased upon a selected pharmacophore monomer.

Either a random or a deterministic combinatorial library can be screenedby the presently disclosed and/or claimed screening methods. In eitherof these two libraries, each unit of the library is isolated and/orimmobilized on a solid support. In the deterministic library, one knowsa priori a particular unit's location on each solid support. In a randomlibrary, the location of a particular unit is not known a priorialthough each site still contains a single unique unit. Many methods forpreparing libraries are known to those of skill in this art (see, e.g.,Geysen et al., Proc. Natl. Acad. Sci. USA, 81:3998–4002 (1984), Houghtenet al., Proc. Natl. Acad. Sci. USA, 81:5131–5135 (1985)). Combinatoriallibrary generated by the any techniques known to those of skill in theart are contemplated (see, e.g., Table 1 of Schultz and Schultz,Biotechnol. Prog., 12(6):729–43 (1996)) for screening; Bartel et al.,Science, 261:1411–1418 (1993); Baumbach et al. BioPharm, (Can):24–35(1992); Bock et al. Nature, 355:564–566 (1992); Borman, S.,Combinatorial chemists focus on samll molecules molecular recognition,and automation, Chem. Eng. News, 2(12):29 (1996); Boublik, et al.,Eukaryotic Virus Display: Engineering the Major Surface Glycoproteins ofthe Autographa California Nuclear Polyhedrosis Virus (ACNPV) for thePresentation of Foreign Proteins on the Virus Surface, Bio/Technology,13:1079–1084 (1995); Brenner, et al., Encoded Combinatorial Chemistry,Proc. Natl. Acad Sci. U.S.A., 89:5381–5383 (1992); Caflisch, et al.,Computational Combinatorial Chemistry for De Novo Ligand Design: Reviewand Assessment, Perspect. Drug Discovery Des., 3:51–84 (1995); Cheng, etal., Sequence-Selective Peptide Binding with aPeptido-A,B-trans-steroidal Receptor Selected from an EncodedCombinatorial Library, J. Am. Chem. Soc., 118:1813–1814 (1996); Chu, etal., Affinity Capillary Electrophoresis to Identify the Peptide in APeptide Library that Binds Most Tightly to Vancomycin, J. Org. Chem.,58:648–652 (1993); Clackson, et al., Making Antibody Fragments UsingPhage Display Libraries, Nature, 352:624–628 (1991); Combs, et al.,Protein Structure-Based Combinatorial Chemistry: Discovery ofNon-Peptide Binding Elements to Src SH3 Domain, J. Am. Chem. Soc.,118:287–288 (1996); Cwirla, et al., Peptides On Phage: A Vast Library ofPeptides for Identifying Ligands, Proc. Natl. Acad. Sci. U.S.A.,87:6378–6382 (1990); Ecker, et al., Combinatorial Drug Discovery: WhichMethod will Produce the Greatest Value, Bio/Technology, 13:351–360(1995); Ellington, et al., In Vitro Selection of RNA Molecules That BindSpecific Ligands, Nature, 346:818–822 (1990); Ellman, J. A., Variants ofBenzodiazephines, J. Am. Chem. Soc., 114:10997 (1992); Erickson, et al.,The Proteins; Neurath, H., Hill, R. L., Eds.: Academic: New York, 1976;pp. 255–257; Felici, et al., J. Mol. 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U.S.A., 92:6419–6423 (1995);Hoogenboom, et al., Multi-Subunit Proteins on the Surface of FilamentousPhage: Methodologies for Displaying Antibody (Fab) Heavy and LightChains, Nucleic Acids Res., 19:4133–4137 (1991); Houghten, et al.,General Method for the Rapid Solid-Phase Synthesis of Large Numbers ofPeptides: Specificity of Antigen-Antibody Interaction at the Level ofIndividual Amino Acids, Proc. Natl. Acad. Sci. U.S.A., 82:5131–5135(1985); Houghten, et al., The Use of Synthetic Peptide CombinatorialLibraries for the Determination of Peptide Ligands in Radio-ReceptorAssays-Opiod-Peptides, Bioorg. Med. Chem. Lett., 3:405–412 (1993);Houghten, et al., Generation and Use of Synthetic Peptide CombinatorialLibraries for Basic Research and Drug Discovery, Nature, 354:84–86(1991); Huang, et al., Discovery of New Ligand Binding Pathways inMyoglobin by Random Mutagenesis, Nature Struct. 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For example, peptides that bind to an MTSP10 polypeptide or a proteasedomain of an SP protein can be identified using phage display libraries.In an exemplary embodiment, this method can include a) contacting phagefrom a phage library with the MTSP10 polypeptide or a protease domainthereof; (b) isolating phage that bind to the protein; and (c)determining the identity of at least one peptide coded by the isolatedphage to identify a peptide that binds to an MTSP10 polypeptide.

H. Modulators of the Activity of MTSP10 Polypeptides

Provided herein are compounds, identified by screening or produced usingthe MTSP10 polypeptide or protease domain in other screening methods,that modulate the activity of an MTSP10. These compounds act by directlyinteracting with the MTSP10 polypeptide or by altering transcription ortranslation thereof. Such molecules include, but are not limited to,antibodies that specifically react with an MTSP10 polypeptide,particularly with the protease domain thereof, antisense nucleic acidsor double-stranded RNA (dsRNA) such as RNAi, that alter expression ofthe MTSP10 polypeptide, antibodies, peptide mimetics and other suchcompounds.

1. Antibodies

Antibodies, including polyclonal and monoclonal antibodies, thatspecifically bind to the MTSP10 polypeptide provided herein,particularly to the single chain protease domains thereof or theactivated forms of the full-length or protease domain or the zymogenform, are provided.

Generally, the antibody is a monoclonal antibody, and typically theantibody specifically binds to the protease domain of the MTSP10polypeptide. In particular embodiments, antibodies to each of the singlechain and or two chain form of the protease domain of MTSP10 areprovided. Also provided are antibodies that specifically bind to anydomain of MTSP10 and to two chain forms thereof.

The MTSP10 polypeptide and domains, fragments, homologs and derivativesthereof can be used as immunogens to generate antibodies thatspecifically bind such immunogens. Such antibodies include but are notlimited to polyclonal, monoclonal, chimeric, single chain, Fabfragments, and an Fab expression library. In a specific embodiment,antibodies to human MTSP10 polypeptide are produced. In anotherembodiment, complexes formed from fragments of an MTSP10 polypeptide,that contain the serine protease domain are used as immunogens forantibody production.

Various procedures known in the art can be used for the production ofpolyclonal antibodies to MTSP10 polypeptide, its domains, derivatives,fragments or analogs. For production of the antibody, various hostanimals can be immunized by injection with the native MTSP10 polypeptideor a synthetic version, or a derivative of the foregoing, such as across-linked MTSP10 polypeptide. Such host animals include but are notlimited to rabbits, mice, rats, etc. Various adjuvants can be used toincrease the immunological response, depending on the host species, andinclude but are not limited to Freund's (complete and incomplete),mineral gels such as aluminum hydroxide, surface active substances suchas lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,dinitrophenol, and potentially useful human adjuvants such as bacilleCalmette-Guerin (BCG) and corynebacterium parvum.

For preparation of monoclonal antibodies directed towards an MTSP10polypeptide or domains, derivatives, fragments or analogs thereof, anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture can be used. Such techniques includebut are not restricted to the hybridoma technique originally developedby Kohler and Milstein (Nature 256:495–497 (1975)), the triomatechnique, the human B-cell hybridoma technique (Kozbor et al.,Immunology Today 4:72 (1983)), and the EBV hybridoma technique toproduce human monoclonal antibodies (Cole et al., in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77–96 (1985)). Inan additional embodiment, monoclonal antibodies can be produced ingerm-free animals utilizing recent technology (PCT/US90/02545). Humanantibodies can be used and can be obtained by using human hybridomas(Cote et al., Proc. Natl. Acad. Sci. USA 80:2026–2030 (1983)), or bytransforming human B cells with EBV virus in vitro (Cole et al., inMonoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77–96(1985)). Techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851–6855(1984); Neuberger et al., Nature 312:604–608 (1984); Takeda et al.,Nature 314:452–454 (1985)) by splicing the genes from a mouse antibodymolecule specific for the MTSP10 polypeptide together with genes from ahuman antibody molecule of appropriate biological activity can be used.

MTSP10-encoding nucleic acid molecules or portions thereof can be usedin DNA immunization protocols to produce antibodies that bind to MTSP10(see, e.g., U.S. Pat. No. 5,795,872 and U.S. Pat. No. 5,643,578 and U.S.Pat. No. 6,337,072).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce MTSP10polypeptide-specific single chain antibodies. An additional embodimentuses the techniques described for the construction of Fab expressionlibraries (Huse et al., Science 246:1275–1281 (1989)) to allow rapid andeasy identification of monoclonal Fab fragments with the desiredspecificity for MTSP10 polypeptide or domains, derivatives, or analogsthereof. Non-human antibodies can be “humanized” by known methods (see,e.g., U.S. Pat. No. 5,225,539).

Antibody fragments that specifically bind to MTSP10 polyeptide orepitopes thereof can be generated by techniques known in the art. Forexample, such fragments include but are not limited to: the F(ab′)2fragment, which can be produced by pepsin digestion of the antibodymolecule; the Fab′ fragments that can be generated by reducing thedisulfide bridges of the F(ab′)2 fragment, the Fab fragments that can begenerated by treating the antibody molecular with papain and a reducingagent, and Fv fragments.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., ELISA(enzyme-linked immunosorbent assay). To select antibodies specific for aparticular domain of the MTSP10 polypeptide one can assay generatedhybridomas for a product that binds to the fragment of the MTSP10polypeptide that contains such a domain.

The foregoing antibodies can be used in methods known in the artrelating to the localization and/or quantitation of MTSP10 polypeptideproteins, e.g., for imaging these proteins, measuring levels thereof inappropriate physiological samples, in, for example, diagnostic methods.In another embodiment, anti-MTSP10 polypeptide antibodies, or fragmentsthereof, containing the binding domain are used as therapeutic agents.

2. Peptides, Polypeptides and Peptide Mimetics

Provided herein are methods for identifying molecules that bind to andmodulate the activity of SP proteins. Included among molecules that bindto SPs, particularly the single chain protease domain or catalyticallyactive fragments thereof, are peptides, polypeptides and peptidemimetics, including cyclic peptides. Peptide mimetics are molecules orcompounds that mimic the necessary molecular conformation of a ligand orpolypeptide for specific binding to a target molecule such as an MTSP10polypeptide. In an exemplary embodiment, the peptides, polypeptides andpeptide mimetics bind to the protease domain of the MTSP10 polypeptide.Such peptides and peptide mimetics include those of antibodies thatspecifically bind to an MTSP10 polypeptide and, typically, bind to theprotease domain of an MTSP10 polypeptide. The peptides, polypeptides andpeptide mimetics identified by methods provided herein can be agonistsor antagonists of MTSP10 polypeptides.

Such peptides, polypeptides and peptide mimetics are useful fordiagnosing, treating, preventing, and screening for a disease ordisorder associated with MTSP10 polypeptide activity in a mammal. Inaddition, the peptides and peptide mimetics are useful for identifying,isolating, and purifying molecules or compounds that modulate theactivity of an MTSP10 polypeptide, or specifically bind to an MTSP10polypeptide, generally the protease domain of an MTSP10 polypeptide. Lowmolecular weight peptides and peptide mimetics can have strong bindingproperties to a target molecule, e.g., an MTSP10 polypeptide or theprotease domain of an MTSP10 polypeptide.

Peptides, polypeptides and peptide mimetics that bind to MTSP10polypeptides as described herein can be administered to mammals,including humans, to modulate MTSP10 polypeptide activity. Thus, methodsfor therapeutic treatment and prevention of neoplastic diseases compriseadministering a peptide, polypeptide or peptide mimetic compound in anamount sufficient to modulate such activity are provided. Thus, alsoprovided herein are methods for treating a subject having such a diseaseor disorder in which a peptide, polypeptide or peptide mimetic compoundis administered to the subject in a therapeutically effective dose oramount.

Compositions containing the peptides, polypeptides or peptide mimeticscan be administered for prophylactic and/or therapeutic treatments. Intherapeutic applications, compositions can be administered to a patientalready suffering from a disease, as described above, in an amountsufficient to cure or at least partially arrest the symptoms of thedisease and its complications. Amounts effective for this use willdepend on the severity of the disease and the weight and general stateof the patient and can be empirically determined.

In prophylactic applications, compositions containing the peptides,polypeptides and peptide mimetics are administered to a patientsusceptible to or otherwise at risk of a particular disease. Such anamount is defined to be a “prophylactically effective dose.” In thisuse, the precise amounts again depend on the patient's state of healthand weight. Accordingly, the peptides, polypeptides and peptide mimeticsthat bind to an MTSP10 polypeptide can be used to prepare pharmaceuticalcompositions containing, as an active ingredient, at least one of thepeptides, polyeptides or peptide mimetics in association with apharmaceutical carrier or diluent. The compounds can be administered,for example, by oral, pulmonary, parental (intramuscular,intraperitoneal, intravenous (IV) or subcutaneous injection), inhalation(via a fine powder formulation), transdermal, nasal, vaginal, rectal, orsublingual routes of administration and can be formulated in dosageforms appropriate for each route of administration (see, e.g.,International PCT application Nos. WO 93/25221 and WO 94/17784; andEuropean Patent Application 613,683).

Peptides, polypeptides and peptide mimetics that bind to MTSP10polypeptides are useful in vitro as unique tools for understanding thebiological role of MTSP10 polypeptides, including the evaluation of themany factors thought to influence, and be influenced by, the productionof MTSP10 polypeptide. Such peptides, polypeptides and peptide mimeticsare also useful in the development of other compounds that bind to andmodulate the activity of an MTSP10 polypeptide, because such compoundsprovide important information on the relationship between structure andactivity that should facilitate such development.

The peptides, polypeptides and peptide mimetics are also useful ascompetitive binders in assays to screen for new MTSP10 polypeptides orMTSP10 polypeptide agonists. In such assay embodiments, the compoundscan be used without modification or can be modified in a variety ofways; for example, by labeling, such as covalently or non-covalentlyjoining a moiety which directly or indirectly provides a detectablesignal. In any of these assays, the materials thereto can be labeledeither directly or indirectly. Possibilities for direct labeling includelabel groups such as: radiolabels such as ¹²⁵I enzymes (U.S. Pat. No.3,645,090) such as peroxidase and alkaline phosphatase, and fluorescentlabels (U.S. Pat. No. 3,940,475) capable of monitoring the change influorescence intensity, wavelength shift, or fluorescence polarization.Possibilities for indirect labeling include biotinylation of oneconstituent followed by binding to avidin coupled to one of the abovelabel groups. The compounds can also include spacers or linkers in caseswhere the compounds are to be attached to a solid support.

Moreover, based on their ability to bind to an MTSP10 polypeptide, thepeptides, polypeptides and peptide mimetics can be used as reagents fordetecting MTSP10 polypeptides in living cells, fixed cells, inbiological fluids, in tissue homogenates and in purified, naturalbiological materials. For example, by labelling such peptides,polypeptides and peptide mimetics, cells having MTSP10 polypeptides canbe identified. In addition, based on their ability to bind an MTSP10polypeptide, the peptides, polypeptides and peptide mimetics can be usedin in situ staining, FACS (fluorescence-activated cell sorting), Westernblotting, ELISA and other analytical protocols. Based on their abilityto bind to an MTSP10 polypeptide, the peptides, polypeptides and peptidemimetics can be used in purification of MTSP10 polypeptides or inpurifying cells expressing the MTSP10 polypeptides, e.g., a polypeptideencoding the protease domain of an MTSP10 polypeptide.

The peptides, polypeptides and peptide mimetics can also be used ascommercial reagents for various medical research and diagnostic uses.The activity of the peptides and peptide mimetics can be evaluatedeither in vitro or in vivo in one of the numerous models described inMcDonald (1992) Am. J. of Pediatric Hematology/Oncology, 14:8–21.

3. Peptide, Polypeptide and Peptide Mimetic Therapy

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compounds are termed “peptidemimetics” or “peptidomimetics” (Luthman et al., A Textbook of DrugDesign and Development, 14:386–406, 2nd Ed., Harwood Academic Publishers(1996); Joachim Grante (1994) Angew. Chem. Int. Ed. Engl., 33:1699–1720;Fauchere (1986) J. Adv. Drug Res., 15:29; Veber and Freidinger (1985)TINS, p. 392; and Evans et al. (1987) J. Med. Chem. 30:1229). Peptidemimetics that are structurally similar to therapeutically usefulpeptides can be used to produce an equivalent or enhanced therapeutic orprophylactic effect. Preparation of peptidomimetics and structuresthereof are known to those of skill in this art.

Systematic substitution of one or more amino acids of a consensussequence with a D-amino acid of the same type (e.g., D-lysine in placeof L-lysine) can be used to generate more stable peptides. In addition,constrained peptides containing a consensus sequence or a substantiallyidentical consensus sequence variation can be generated by methods knownin the art (Rizo et al. (1992) An. Rev. Biochem., 61:387, incorporatedherein by reference); for example, by adding internal cysteine residuescapable of forming intramolecular disulfide bridges which cyclize thepeptide.

Those skilled in the art appreciate that modifications can be made tothe peptides and mimetics without deleteriously effecting the biologicalor functional activity of the peptide. Further, the skilled artisanwould know how to design non-peptide structures in three dimensionalterms, that mimic the peptides that bind to a target molecule, e.g., anMTSP10 polypeptide or, generally, the protease domain of MTSP10polypeptides (see, e.g., Eck and Sprang (1989) J. Biol. Chem., 26:17605–18795).

When used for diagnostic purposes, the peptides and peptide mimetics canbe labeled with a detectable label and, accordingly, the peptides andpeptide mimetics without such a label can serve as intermediates in thepreparation of labeled peptides and peptide mimetics. Detectable labelscan be molecules or compounds, which when covalently attached to thepeptides and peptide mimetics, permit detection of the peptide andpeptide mimetics in vivo, for example, in a patient to whom the peptideor peptide mimetic has been administered, or in vitro, e.g., in a sampleor cells. Suitable detectable labels are well known in the art andinclude, by way of example, radioisotopes, fluorescent labels (e.g.,fluorescein), and the like. The particular detectable label employed isnot critical and is selected to be detectable at non-toxic levels.Selection of the such labels is well known in the art.

Covalent attachment of a detectable label to the peptide or peptidemimetic is accomplished by conventional methods well known in the art.For example, when the ¹²⁵I radioisotope is employed as the detectablelabel, covalent attachment of ¹²⁵I to the peptide or the peptide mimeticcan be achieved by incorporating the amino acid tyrosine into thepeptide or peptide mimetic and then iodinating the peptide (see, e.g.,Weaner et al. (1994) Synthesis and Applications of Isotopically LabelledCompounds, pp. 137–140). If tyrosine is not present in the peptide orpeptide mimetic, incorporation of tyrosine to the N or C terminus of thepeptide or peptide mimetic can be achieved by well known chemistry.Likewise, ³²P can be incorporated onto the peptide or peptide mimetic asa phosphate moiety through, for example, a hydroxyl group on the peptideor peptide mimetic using conventional chemistry.

Labeling of peptidomimetics usually involves covalent attachment of oneor more labels, directly or through a spacer (e.g., an amide group), tonon-interfering position(s) on the peptidomimetic that are predicted byquantitative structure-activity data and/or molecular modeling. Suchnon-interfering positions generally are positions that do not formdirect contacts with the macromolecules(s) to which the peptidomimeticbinds to produce the therapeutic effect. Derivatization (e.g., labeling)of peptidomimetics should not substantially interfere with the desiredbiological or pharmacological activity of the peptidomimetic.

Peptides, polypeptides and peptide mimetics that can bind to an MTSP10polypeptide or the protease domain of MTSP10 polypeptides and/ormodulate the activity thereof, or exhibit MTSP10 polypeptide activity,can be used for treatment of neoplastic disease. The peptides,polypeptides and peptide mimetics can be delivered, in vivo or ex vivo,to the cells of a subject in need of treatment. Further, peptides whichhave MTSP10 polypeptide activity can be delivered, in vivo or ex vivo,to cells which carry mutant or missing alleles encoding the MTSP10polypeptide gene. Any of the techniques described herein or known to theskilled artisan can be used for preparation and in vivo or ex vivodelivery of such peptides, polypeptides and peptide mimetics that aresubstantially free of other human proteins. For example, the peptides,polypeptides and peptide mimetics can be readily prepared by expressionin a microorganism or synthesis in vitro.

The peptides or peptide mimetics can be introduced into cells, in vivoor ex vivo, by microinjection or by use of liposomes, for example.Alternatively, the peptides, polypeptides or peptide mimetics can betaken up by cells, in vivo or ex vivo, actively or by diffusion. Inaddition, extracellular application of the peptide, polypeptide orpeptide mimetic can be sufficient to effect treatment of a neoplasticdisease. Other molecules, such as drugs or organic compounds, that: 1)bind to a MTSP10 polypeptide or protease domain thereof; or 2) have asimilar function or activity to an MTSP10 polypeptide or protease domainthereof, can be used in methods for treatment.

4. Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptides or peptides of interest or of smallmolecules or peptide mimetics with which they interact (e.g., agonistsand antagonists) in order to fashion drugs which are, e.g., more activeor stable forms thereof; or which, for example, enhance or interferewith the function of a polypeptide in vivo (e.g., an MTSP10polypeptide). In one approach, one first determines thethree-dimensional structure of a protein of interest (e.g., an MTSP10polypeptide or polypeptide having a protease domain) or, for example, ofan MTSP10 polypeptide-ligand complex, by X-ray crystallography, bycomputer modeling or most typically, by a combination of approaches(see, e.g., Erickson et al. 1990). Also, useful information regardingthe structure of a polypeptide can be gained by modeling based on thestructure of homologous proteins. In addition, peptides can be analyzedby an alanine scan. In this technique, an amino acid residue is replacedby Ala, and its effect on the peptide's activity is determined. Each ofthe amino acid residues of the peptide is analyzed in this manner todetermine the important regions of the peptide.

Also, a polypeptide or peptide that binds to an MTSP10 polypeptide or,generally, the protease domain of an MTSP10 polypeptide, can be selectedby a functional assay, and then the crystal structure of thispolypeptide or peptide can be determined. The polypeptide can be, forexample, an antibody specific for an MTSP10 polypeptide or the proteindomain of an MTSP10 polypeptide. This approach can yield a pharmacophoreupon which subsequent drug design can be based. Further, it is possibleto bypass the crystallography altogether by generating anti-idiotypicpolypeptides or peptides, (anti-ids) to a functional, pharmacologicallyactive polypeptide or peptide that binds to an MTSP10 polypeptide orprotease domain of an MTSP10 polypeptide. As a mirror image of a mirrorimage, the binding site of the anti-ids is expected to be an analog ofthe original target molecule, e.g., an MTSP10 polypeptide or polypeptidehaving an MTSP10 polypeptide. The anti-id could then be used to identifyand isolate peptides from banks of chemically or biologically producedbanks of peptides. Selected peptides would then act as thepharmacophore.

Thus, one can design drugs which have, for example, improved activity orstability or which act as modulators (e.g., inhibitors, agonists orantagonists) of MTSP10 polypeptide activity, and are useful in themethods, particularly the methods for diagnosis, treatment, prevention,and screening of a neoplastic disease. By virtue of the availability ofnucleic acid that encodes MTSP10 polypeptides, sufficient amounts of theMTSP10 polypeptide can be made available to perform such analyticalstudies as X-ray crystallography. In addition, the knowledge of theamino acid sequence of an MTSP10 polypeptide or the protease domainthereof, e.g., the protease domain encoded by the amino acid sequence ofSEQ ID Nos. 5 and 6, can provide guidance on computer modelingtechniques in place of, or in addition to, X-ray crystallography.

Methods of Identifying Peptides and Peptide Mimetics that Bind to MTSP10Polypeptides

Peptides having a binding affinity to the MTSP10 polypeptide providedherein (e.g., an MTSP10 polypeptide or a polypeptide having a proteasedomain of an MTSP10 polypeptide) can be readily identified, for example,by random peptide diversity generating systems coupled with an affinityenrichment process. Specifically, random peptide diversity generatingsystems include the “peptides on plasmids” system (see, e.g., U.S. Pat.Nos. 5,270,170 and 5,338,665); the “peptides on phage” system (see,e.g., U.S. Pat. No. 6,121,238 and Cwirla, et al. (1990) Proc. Natl.Acad. Sci. U.S.A. 87:6378–6382); the “polysome system;” the “encodedsynthetic library (ESL)” system; and the “very large scale immobilizedpolymer synthesis” system (see, e.g., U.S. Pat. No. 6,121,238; and Doweret al. (1991) An. Rep. Med. Chem. 26:271–280

For example, using the procedures described above, random peptides cangenerally be designed to have a defined number of amino acid residues inlength (e.g., 12). To generate the collection of oligonucleotidesencoding the random peptides, the codon motif (NNK)x, where N isnucleotide A, C, G, or T (equimolar; depending on the methodologyemployed, other nucleotides can be employed), K is G or T (equimolar),and x is an integer corresponding to the number of amino acids in thepeptide (e.g., 12) can be used to specify any one of the 32 possiblecodons resulting from the NNK motif: 1 for each of 12 amino acids, 2 foreach of 5 amino acids, 3 for each of 3 amino acids, and only one of thethree stop codons. Thus, the NNK motif encodes all of the amino acids,encodes only one stop codon, and reduces codon bias.

The random peptides can be presented, for example, either on the surfaceof a phage particle, as part of a fusion protein containing either thepill or the pVIII coat protein of a phage fd derivative (peptides onphage) or as a fusion protein with the LacI peptide fusion protein boundto a plasmid (peptides on plasmids). The phage or plasmids, includingthe DNA encoding the peptides, can be identified and isolated by anaffinity enrichment process using immobilized MTSP10 polypeptide havinga protease domain. The affinity enrichment process, sometimes called“panning,” typically involves multiple rounds of incubating the phage,plasmids, or polysomes with the immobilized MTSP10 polypeptide,collecting the phage, plasmids, or polysomes that bind to the MTSP10polypeptide (along with the accompanying DNA or mRNA), and producingmore of the phage or plasmids (along with the accompanying LacI-peptidefusion protein) collected.

Characteristics of Peptides and Peptide Mimetics

Among the peptides, polypeptides and peptide mimetics for therapeuticapplication are those of having molecular weights from about 250 toabout 8,000 daltons. If such peptides are oligomerized, dimerized and/orderivatized with a hydrophilic polymer (e.g., to increase the affinityand/or activity of the compounds), the molecular weights of suchpeptides can be substantially greater and can range anywhere from about500 to about 120,000 daltons, generally from about 8,000 to about 80,000daltons. Such peptides can contain 9 or more amino acids that arenaturally occurring or synthetic (non-naturally occurring) amino acids.One skilled in the art can determine the affinity and molecular weightof the peptides and peptide mimetics suitable for therapeutic and/ordiagnostic purposes (e.g., see Dower et al., U.S. Pat. No. 6,121,238).

The peptides can be covalently attached to one or more of a variety ofhydrophilic polymers. Suitable hydrophilic polymers include, but are notlimited to, polyalkylethers as exemplified by polyethylene glycol andpolypropylene glycol, polylactic acid, polyglycolic acid,polyoxyalkenes, polyvinylalcohol, polyvinylpyrrolidone, cellulose andcellulose derivatives, dextran and dextran derivatives. When the peptidecompounds are derivatized with such polymers, their solubility andcirculation half-lives can be increased with little, if any,diminishment in their binding activity. The peptide compounds can bedimerized and each of the dimeric subunits can be covalently attached toa hydrophilic polymer. The peptide compounds can be PEGylated, i.e.,covalently attached to polyethylene glycol (PEG).

5. Methods of Preparing Peptides and Peptide Mimetics

Peptides that bind to MTSP10 polypeptides can be prepared by classicalmethods known in the art, for example, by using standard solid phasetechniques. The standard methods include exclusive solid phasesynthesis, partial solid phase synthesis methods, fragment condensation,classical solution synthesis, and even by recombinant DNA technology(see, e.g., Merrifield (1963) J. Am. Chem. Soc., 85:2149, incorporatedherein by reference.)

Using the “encoded synthetic library” or “very large scale immobilizedpolymer synthesis” systems (see, e.g., U.S. Pat. Nos. 5 ,925,525, and5,902,723), the minimum size of a peptide with the activity of interestcan be determined. In addition all peptides that form the group ofpeptides that differ from the desired motif (or the minimum size of thatmotif) in one, two, or more residues can be prepared. This collection ofpeptides then can be screened for the ability to bind to the targetmolecule, e.g., MTSP10 polypeptide or, generally, the protease domain ofan MTSP10 polypeptide. This immobilized polymer synthesis system orother peptide synthesis methods can also be used to synthesizetruncation analogs and deletion analogs and combinations of truncationand deletion analogs of the peptide compounds.

These procedures can also be used to synthesize peptides in which aminoacids other than the 20 naturally occurring, genetically encoded aminoacids are substituted at one, two, or more positions of the peptide. Forinstance, naphthylalanine can be substituted for tryptophan,facilitating synthesis. Other synthetic amino acids that can besubstituted into the peptides include L-hydroxypropyl,L-3,4-dihydroxy-phenylalanyl, d amino acids such as L-d-hydroxylysyl andD-d-methylalanyl, L-α-methylalanyl, β amino acids, and isoquinolyl. Damino acids and non-naturally occurring synthetic amino acids can alsobe incorporated into the peptides (see, e.g., Roberts et al. (1983)Unusual Amino/Acids in Peptide Synthesis, 5(6):341–449).

The peptides also can be modified by phosphorylation (see, e.g., W.Bannwarth et al. (1996) Biorganic and Medicinal Chemistry Letters,6(17):2141–2146), and other methods for making peptide derivatives (see,e.g., Hruby et al. (1990) Biochem. J., 268(2):249–262). Thus, peptidecompounds also serve as a basis to prepare peptide mimetics with similaror improved biological activity.

Those of skill in the art recognize that a variety of techniques areavailable for constructing peptide mimetics with the same or similardesired biological activity as the corresponding peptide compound butwith more favorable activity than the peptide with respect tosolubility, stability, and susceptibility to hydrolysis and proteolysis(see, e.g., Morgan et al. (1989) An. Rep. Med. Chem., 24:243–252).Methods for preparing peptide mimetics modified at the N-terminal aminogroup, the C-terminal carboxyl group, and/or changing one or more of theamido linkages in the peptide to a non-amido linkage are known to thoseof skill in the art.

Amino terminus modifications include, but are not limited to,alkylating, acetylating and adding a carbobenzoyl group, forming asuccinimide group (see, e.g., Murray et al. (1995) Burger's MedicinalChemistry and Drug Discovery, 5th ed., Vol. 1, Manfred E. Wolf, ed.,John Wiley and Sons, Inc.). C-terminal modifications include mimeticswherein the C-terminal carboxyl group is replaced by an ester, an amideor modifications to form a cyclic peptide.

In addition to N-terminal and C-terminal modifications, the peptidecompounds, including peptide mimetics, can advantageously be modifiedwith or covalently coupled to one or more of a variety of hydrophilicpolymers. It has been found that when peptide compounds are derivatizedwith a hydrophilic polymer, their solubility and circulation half-livescan be increased and their immunogenicity is masked, with little, ifany, diminishment in their binding activity. Suitable nonproteinaceouspolymers include, but are not limited to, polyalkylethers as exemplifiedby polyethylene glycol and polypropylene glycol, polylactic acid,polyglycolic acid, polyoxyalkenes, polyvinylalcohol,polyvinylpyrrolidone, cellulose and cellulose derivatives, dextran anddextran derivatives. Generally, such hydrophilic polymers have anaverage molecular weight ranging from about 500 to about 100,000daltons, including from about 2,000 to about 40,000 daltons and, fromabout 5,000 to about 20,000 daltons. The hydrophilic polymers also canhave an average molecular weights of about 5,000 daltons, 10,000 daltonsand 20,000 daltons.

Methods for derivatizing peptide compounds or for coupling peptides tosuch polymers have been described (see, e.g., Zallipsky (1995)Bioconjugate Chem., 6:150–165; Monfardini et al. (1995) BioconjugateChem., 6:62–69; U.S. Pat. No. 4,640,835; U.S. Pat. No. 4,496,689; U.S.Pat. No. 4,301,144; U.S. Pat. No. 4,670,417; U.S. Pat. No. 4,791,192;U.S. Pat. No. 4,179,337 and WO 95/34326, all of which are incorporatedby reference in their entirety herein).

Other methods for making peptide derivatives are described, for example,in Hruby et al. (1990), Biochem J., 268(2):249–262, which isincorporated herein by reference. Thus, the peptide compounds also serveas structural models for non-peptidic compounds with similar biologicalactivity. Those of skill in the art recognize that a variety oftechniques are available for constructing compounds with the same orsimilar desired biological activity as a particular peptide compound butwith more favorable activity with respect to solubility, stability, andsusceptibility to hydrolysis and proteolysis (see, e.g., Morgan et al.(1989) An. Rep. Med. Chem., 24:243–252, incorporated herein byreference). These techniques include replacing the peptide backbone witha backbone composed of phosphonates, amidates, carbamates, sulfonamides,secondary amines, and N-methylamino acids.

Peptide compounds can exist in a cyclized form with an intramoleculardisulfide bond between the thiol groups of the cysteines. Alternatively,an intermolecular disulfide bond between the thiol groups of thecysteines can be produced to yield a dimeric (or higher oligomeric)compound. One or more of the cysteine residues can also be substitutedwith a homocysteine.

I. Conjugates

A conjugate, containing: a) a single chain protease domain (orproteolytically active portion thereof) of an MTSP10 polypeptide or afull length zymogen, activated form thereof, or two or single chainprotease domain thereof; and b) a targeting agent linked to the MTSP10polypeptide directly or via a linker, wherein the agent facilitates: i)affinity isolation or purification of the conjugate; ii) attachment ofthe conjugate to a surface; iii) detection of the conjugate; or iv)targeted delivery to a selected tissue or cell, is provided herein. Theconjugate can be a chemical conjugate or a fusion protein mixturethereof.

The targeting agent can be a protein or peptide fragment, such as atissue specific or tumor specific monoclonal antibody or growth factoror fragment thereof linked either directly or via a linker to an MTSP10polypeptide or a protease domain thereof. The targeting agent can alsobe a protein or peptide fragment that contains a protein bindingsequence, a nucleic acid binding sequence, a lipid binding sequence, apolysaccharide binding sequence, or a metal binding sequence, or alinker for attachment to a solid support. In a particular embodiment,the conjugate contains a) the MTSP10 or portion thereof, as describedherein; and b) a targeting agent linked to the MTSP10 polypeptidedirectly or via a linker.

Conjugates, such as fusion proteins and chemical conjugates, of theMTSP10 polypeptide with a protein or peptide fragment (or pluralitythereof) that functions, for example, to facilitate affinity isolationor purification of the MTSP10 polypeptide domain, attachment of theMTSP10 polypeptide domain to a surface, or detection of the MTSP10polypeptide domain are provided. The conjugates can be produced bychemical conjugation, such as via thiol linkages, and can be produced byrecombinant means as fusion proteins. In the fusion protein, the peptideor fragment thereof is linked to either the N-terminus or C-terminus ofthe MTSP10 polypeptide domain. In chemical conjugates the peptide orfragment thereof can be linked anywhere that conjugation can beeffected, and there can be a plurality of such peptides or fragmentslinked to a single MTSP10 polypeptide domain or to a plurality thereof.

The targeting agent is for in vitro or in vivo delivery to a cell ortissue, and includes agents such as cell or tissue-specific antibodies,growth factors and other factors (including compounds) that bind tomoieties expressed on specific cells; and other cell or tissue specificagents that promote directed delivery of a linked protein. The targetingagent can be one that specifically delivers the MTSP10 polypeptide toselected cells by interaction with a cell surface protein andinternalization of conjugate or MTSP10 polypeptide portion thereof.

These conjugates are used in a variety of methods and are particularlysuited for use in methods of activation of prodrugs, such as prodrugsthat upon cleavage by the particular MTSP10, which is localized at ornear the targeted cell or tissue, are cytotoxic. The prodrugs areadministered prior to, or simultaneously with, or subsequently to theconjugate. Upon delivery to the targeted cells, the protease activatesthe prodrug, which then exhibits a therapeutic effect, such as acytotoxic effect.

1. Conjugation

Conjugates with linked MTSP10 polypeptide domains can be prepared eitherby chemical conjugation, recombinant DNA technology, or combinations ofrecombinant expression and chemical conjugation. The MTSP10 polypeptidedomains and the targeting agent can be linked in any orientation andmore than one targeting agents and/or MTSP10 polypeptide domains can bepresent in a conjugate.

a. Fusion Proteins

Fusion proteins are provided herein. A fusion protein contains: a) oneor a plurality of domains of an MTSP10 polypeptide and b) a targetingagent. The fusion proteins are generally produced by recombinantexpression of nucleic acids that encode the fusion protein.

b. Chemical Conjugation

To effect chemical conjugation herein, the MTSP10 polypeptide domain islinked via one or more selected linkers or directly to the targetingagent. Chemical conjugation must be used if the targeted agent is otherthan a peptide or protein, such as a nucleic acid or a non-peptide drug.Any means known to those of skill in the art for chemically conjugatingselected moieties can be used.

2. Linkers

Linkers for two purposes are contemplated herein. The conjugates caninclude one or more linkers between the MTSP10 polypeptide portion andthe targeting agent. Additionally, linkers are used for facilitating orenhancing immobilization of an MTSP10 polypeptide or portion thereof ona solid support, such as a microtiter plate, silicon or silicon-coatedchip, glass or plastic support, such as for high throughput solid phasescreening protocols.

Any linker known to those of skill in the art for preparation ofconjugates can be used herein. These linkers are typically used in thepreparation of chemical conjugates; peptide linkers can be incorporatedinto fusion proteins.

Linkers can be any moiety suitable to associate a domain of MTSP10polypeptide and a targeting agent. Such linkers and linkages include,but are not limited to, peptidic linkages, amino acid and peptidelinkages, typically containing between one and about 60 amino acids,more generally between about 10 and 30 amino acids, chemical linkers,such as heterobifunctional cleavable cross-linkers, including but arenot limited to, N-succinimidyl (4-iodoacetyl)-aminobenzoate,sulfosuccinimydil (4-iodoacetyl)-aminobenzoate,4-succinimidyl-oxycarbonyl-a- (2-pyridyidithio)toluene,sulfosuccinimidyl-6-[a-methyl-a-(pyridyldithiol)-toluamido]hexanoate,N-succinimidyl-3-(-2-pyridyidithio)-proprionate, succinimidyl6[3(-(-2-pyridyidithio)-proprionamido] hexanoate, sulfosuccinimidyl6[3(-(-2-pyridyidithio)-propionamido] hexanoate,3-(2-pyridyidithio)-propionyl hydrazide, Ellman's reagent,dichlorotriazinic acid, and S-(2-thiopyridyl)-L-cysteine. Other linkersinclude, but are not limited to peptides and other moieties that reducestearic hindrance between the domain of MTSP10 polypeptide and thetargeting agent, intracellular enzyme substrates, linkers that increasethe flexibility of the conjugate, linkers that increase the solubilityof the conjugate, linkers that increase the serum stability of theconjugate, photocleavable linkers and acid cleavable linkers.

Other exemplary linkers and linkages that are suitable for chemicallylinked conjugates include, but are not limited to, disulfide bonds,thioether bonds, hindered disulfide bonds, and covalent bonds betweenfree reactive groups, such as amine and thiol groups. These bonds areproduced using heterobifunctional reagents to produce reactive thiolgroups on one or both of the polypeptides and then reacting the thiolgroups on one polypeptide with reactive thiol groups or amine groups towhich reactive maleimido groups or thiol groups can be attached on theother. Other linkers include, acid cleavable linkers, such asbismaleimideothoxy propane, acid labile-transferrin conjugates andadipic acid diihydrazide, that would be cleaved in more acidicintracellular compartments; cross linkers that are cleaved upon exposureto UV or visible light; and linkers, such as various domains, such asC_(H)1, C_(H)2, and C_(H)3, from the constant region of human IgG₁ (see,Batra et al. Molecular Immunol., 30:379–386 (1993)). In someembodiments, several linkers can be included in order to take advantageof desired properties of each linker.

Chemical linkers and peptide linkers can be inserted by covalentlycoupling the linker to the domain of MTSP10 polypeptide and thetargeting agent. The heterobifunctional agents, described below, can beused to effect such covalent coupling. Peptide linkers can also belinked by expressing DNA encoding the linker and therapeutic agent (TA),linker and targeted agent, or linker, targeted agent and therapeuticagent (TA) as a fusion protein. Flexible linkers and linkers thatincrease solubility of the conjugates are contemplated for use, eitheralone or with other linkers are also contemplated herein.

a) Acid Cleavable, Photocleavable and Heat Sensitive Linkers

Acid cleavable linkers, photocleavable and heat sensitive linkers canalso be used, particularly where it can be necessary to cleave thedomain of MTSP10 polypeptide to permit it to be more readily accessibleto reaction. Acid cleavable linkers include, but are not limited to,bismaleimideothoxy propane; and adipic acid dihydrazide linkers (see,e.g., Fattom et al. (1992) Infection & Immun. 60:584–589) and acidlabile transferrin conjugates that contain a sufficient portion oftransferrin to permit entry into the intracellular transferrin cyclingpathway (see, e.g., Welhöner et al. (1991) J. Biol. Chem.266:4309–4314).

Photocleavable linkers are linkers that are cleaved upon exposure tolight (see, e.g., Goldmacher et al. (1992) Bioconj. Chem. 3:104–107,which linkers are herein incorporated by reference), thereby releasingthe targeted agent upon exposure to light. Photocleavable linkers thatare cleaved upon exposure to light are known (see, e.g., Hazum et al.(1981) in Pept., Proc. Eur. Pept. Symp., 16th, Brunfeldt, K (Ed), pp.105–110, which describes the use of a nitrobenzyl group as aphotocleavable protective group for cysteine; Yen et al. (1989)Makromol. Chem 190:69–82, which describes water soluble photocleavablecopolymers, including hydroxypropylmethacrylamide copolymer, glycinecopolymer, fluorescein copolymer and methylrhodamine copolymer;Goldmacher et al. (1992) Bioconj. Chem. 3:104–107, which describes across-linker and reagent that undergoes photolytic degradation uponexposure to near UV light (350 nm); and Senter et al. (1985) Photochem.Photobiol 42:231–237, which describes nitrobenzyloxycarbonyl chloridecross linking reagents that produce photocleavable linkages), therebyreleasing the targeted agent upon exposure to light. Such linkers wouldhave particular use in treating dermatological or ophthalmic conditionsthat can be exposed to light using fiber optics. After administration ofthe conjugate, the eye or skin or other body part can be exposed tolight, resulting in release of the targeted moiety from the conjugate.Such photocleavable linkers are useful in connection with diagnosticprotocols in which it is desirable to remove the targeting agent topermit rapid clearance from the body of the animal.

b) Other Linkers for Chemical Conjugation

Other linkers, include trityl linkers, particularly, derivatized tritylgroups to generate a genus of conjugates that provide for release oftherapeutic agents at various degrees of acidity or alkalinity. Theflexibility thus afforded by the ability to preselect the pH range atwhich the therapeutic agent is released allows selection of a linkerbased on the known physiological differences between tissues in need ofdelivery of a therapeutic agent (see, e.g., U.S. Pat. No. 5,612,474).For example, the acidity of tumor tissues appears to be lower than thatof normal tissues.

c) Peptide Linkers

The linker moieties can be peptides. Peptide linkers can be employed infusion proteins and also in chemically linked conjugates. The peptidetypically has from about 2 to about 60 amino acid residues, for examplefrom about 5 to about 40, or from about 10 to about 30 amino acidresidues. The length selected depends upon factors, such as the use forwhich the linker is included.

Peptide linkers are advantageous when the targeting agent isproteinaceous. For example, the linker moiety can be a flexible spaceramino acid sequence, such as those known in single-chain antibodyresearch. Examples of such known linker moieties include, but are notlimited to, peptides, such as (Gly_(m)Ser)_(n) and (Ser_(m)Gly)_(n), inwhich n is 1 to 6, including 1 to 4 and 2 to 4, and m is 1 to 6,including 1 to 4, and 2 to 4, enzyme cleavable linkers and others.

Additional linking moieties are described, for example, in Huston etal., Proc. Natl. Acad. Sci. U.S.A. 85:5879–5883, 1988; Whitlow, M., etal., Protein Engineering 6:989–995, 1993; Newton et al., Biochemistry35:545–553, 1996; A. J. Cumber et al., Bioconj. Chem. 3:397–401, 1992;Ladurner et al., J. Mol. Biol. 273:330–337, 1997; and U.S. Pat. No.4,894,443. In some embodiments, several linkers can be included in orderto take advantage of desired properties of each linker.

3. Targeting Agents

Any agent that facilitates detection, immobilization, or purification ofthe conjugate is contemplated for use herein. For chemical conjugatesany moiety that has such properties is contemplated; for fusionproteins, the targeting agent is a protein, peptide or fragment thereofthat is sufficient to effects the targeting activity. Contemplatedtargeting agents include those that deliver the MTSP10 polypeptide orportion thereof to selected cells and tissues. Such agents include tumorspecific monoclonal antibodies and portions thereof, growth factors,such as FGF, EGF, PDGF, VEGF, cytokines, including chemokines, and othersuch agents.

4. Nucleic Acids, Plasmids and Cells

Isolated nucleic acid fragments encoding fusion proteins are provided.The nucleic acid fragment that encodes the fusion protein includes: a)nucleic acid encoding a protease domain of an MTSP10 polypeptide; and b)nucleic acid encoding a protein, peptide or effective fragment thereofthat facilitates: i) affinity isolation or purification of the fusionprotein; ii) attachment of the fusion protein to a surface; or iii)detection of the fusion protein. Generally, the nucleic acid is DNA.

Plasmids for replication and vectors for expression that contain theabove nucleic acid fragments are also provided. Cells containing theplasmids and vectors are also provided. The cells can be any suitablehost including, but are not limited to, bacterial cells, yeast cells,fungal cells, plant cells, insect cell and animal cells. The nucleicacids, plasmids, and cells containing the plasmids can be preparedaccording to methods known in the art including any described herein.

Also provided are methods for producing the above fusion proteins. Anexemplary method includes the steps of growing, for example, culturingthe cells so that they proliferate, cells containing a plasmid encodingthe fusion protein under conditions whereby the fusion protein isexpressed by the cell, and recovering the expressed fusion protein.Methods for expressing and recovering recombinant proteins are wellknown in the art (See generally, Current Protocols in Molecular Biology(1998) § 16, John Wiley & Sons, Inc.) and such methods can be used forexpressing and recovering the expressed fusion proteins.

The recovered fusion proteins can be isolated or purified by methodsknown in the art such as centrifugation, filtration, chromatography,electrophoresis, immunoprecipitation, and other such methods, or by acombination thereof (See generally, Current Protocols in MolecularBiology (1998) § 10, John Wiley & Sons, Inc.). Generally the recoveredfusion protein is isolated or purified through affinity binding betweenthe protein or peptide fragment of the fusion protein and an affinitybinding moiety. As discussed in the above sections regarding theconstruction of the fusion proteins, any affinity binding pairs can beconstructed and used in the isolation or purification of the fusionproteins. For example, the affinity binding pairs can be protein bindingsequences/protein, DNA binding sequences/DNA sequences, RNA bindingsequences/RNA sequences, lipid binding sequences/lipid, polysaccharidebinding sequences/polysaccharide, or metal binding sequences/metal.

5. Immobilization and Supports or Substrates Therefor

In certain embodiments, where the targeting agents are designed forlinkage to surfaces, the MTSP10 polypeptide can be attached by linkagesuch as ionic or covalent, non-covalent or other chemical interaction,to a surface of a support or matrix material. Immobilization can beeffected directly or via a linker. The MTSP10 polypeptide can beimmobilized on any suitable support, including, but are not limited to,silicon chips, and other supports described herein and known to those ofskill in the art. A plurality of MTSP10 polypeptide or protease domainsthereof can be attached to a support, such as an array (i.e., a patternof two or more) of conjugates on the surface of a silicon chip or otherchip for use in high throughput protocols and formats.

It is also noted that the domains of the MTSP10 polypeptide can belinked directly to the surface or via a linker without a targeting agentlinked thereto. Hence chips containing arrays of the domains of theMTSP10 polypeptide.

The matrix material or solid supports contemplated herein are generallyany of the insoluble materials known to those of skill in the art toimmobilize ligands and other molecules, and are those that used in manychemical syntheses and separations. Such supports are used, for example,in affinity chromatography, in the immobilization of biologically activematerials, and during chemical syntheses of biomolecules, includingproteins, amino acids and other organic molecules and polymers. Thepreparation of and use of supports is well known to those of skill inthis art; there are many such materials and preparations thereof known.For example, naturally-occurring support materials, such as agarose andcellulose, can be isolated from their respective sources, and processedaccording to known protocols, and synthetic materials can be prepared inaccord with known protocols.

The supports are typically insoluble materials that are solid, porous,deformable, or hard, and have any required structure and geometry,including, but not limited to: beads, pellets, disks, capillaries,hollow fibers, needles, solid fibers, random shapes, thin films andmembranes. Thus, the item can be fabricated from the matrix material orcombined with it, such as by coating all or part of the surface orimpregnating particles.

Typically, when the matrix is particulate, the particles are at leastabout 10–2000 μm, but can be smaller or larger, depending upon theselected application. Selection of the matrices is governed, at least inpart, by their physical and chemical properties, such as solubility,functional groups, mechanical stability, surface area swellingpropensity, hydrophobic or hydrophilic properties and intended use.

If necessary, the support matrix material can be treated to contain anappropriate reactive moiety. In some cases, the support matrix materialalready containing the reactive moiety can be obtained commercially. Thesupport matrix material containing the reactive moiety can thereby serveas the matrix support upon which molecules are linked. Materialscontaining reactive surface moieties such as amino silane linkages,hydroxyl linkages or carboxysilane linkages can be produced by wellestablished surface chemistry techniques involving silanizationreactions, or the like. Examples of these materials are those havingsurface silicon oxide moieties, covalently linked togamma-aminopropyl-silane, and other organic moieties;N-[3-(triethyoxysilyl)propyl]phthelamic acid; andbis-(2-hydroxyethyl)aminopropyltriethoxysilane. Exemplary of readilyavailable materials containing amino group reactive functionalities,include, but are not limited to, para-aminophenyltriethyoxysilane. Alsoderivatized polystyrenes and other such polymers are well known andreadily available to those of skill in this art (e.g., the Tentagel®Resins are available with a multitude of functional groups, and are soldby Rapp Polymere, Tubingen, Germany; see, U.S. Pat. No. 4,908,405 andU.S. Pat. No. 5,292,814; see, also Butz et al., Peptide Res., 7:20–23(1994); and Kleine et al., Immunobiol., 190:53–66 (1994)).

These matrix materials include any material that can act as a supportmatrix for attachment of the molecules of interest. Such materials areknown to those of skill in this art, and include those that are used asa support matrix. These materials include, but are not limited to,inorganics, natural polymers, and synthetic polymers, including, but arenot limited to: cellulose, cellulose derivatives, acrylic resins, glass,silica gels, polystyrene, gelatin, polyvinyl pyrrolidone, co-polymers ofvinyl and acrylamide, polystyrene cross-linked with divinylbenzene andothers (see, Merrifield, Biochemistry, 3:1385–1390 (1964)),polyacrylamides, latex gels, polystyrene, dextran, polyacrylamides,rubber, silicon, plastics, nitrocellulose, celluloses, natural sponges.Of particular interest herein, are highly porous glasses (see, e.g.,U.S. Pat. No. 4,244,721) and others prepared by mixing a borosilicate,alcohol and water.

Synthetic supports include, but are not limited to: acrylamides,dextran-derivatives and dextran co-polymers, agarose-polyacrylamideblends, other polymers and co-polymers with various functional groups,methacrylate derivatives and co-polymers, polystyrene and polystyrenecopolymers (see, e.g., Merrifield, Biochemistry, 3:1385–1390 (1964);Berg et al., in Innovation Perspect. Solid Phase Synth. Collect. Pap.,Int. Symp., 1st, Epton, Roger (Ed), pp. 453–459 (1990); Berg et al.,Pept., Proc. Eur. Pept. Symp., 20th, Jung, G. et al. (Eds), pp. 196–198(1989); Berg et al., J. Am. Chem. Soc., 111:8024–8026 (1989); Kent etal., Isr. J. Chem., 17:243–247 (1979); Kent et al., J. Org. Chem.,43:2845–2852 (1978); Mitchell et al., Tetrahedron Lett., 42:3795–3798(1976); U.S. Pat. No. 4,507,230; U.S. Pat. No. 4,006,117; and U.S. Pat.No. 5,389,449). Such materials include those made from polymers andco-polymers such as polyvinylalcohols, acrylates and acrylic acids suchas polyethylene-co-acrylic acid, polyethylene-co-methacrylic acid,polyethylene-co-ethylacrylate, polyethylene-co-methyl acrylate,polypropylene-co-acrylic acid, polypropylene-co-methyl-acrylic acid,polypropylene-co-ethyl-acrylate, polypropylene-co-methyl acrylate,polyethylene-co-vinyl acetate, polypropylene-co-vinyl acetate, and thosecontaining acid anhydride groups such as polyethylene-co-maleicanhydride and polypropylene-co-maleic anhydride. Liposomes have alsobeen used as solid supports for affinity purifications (Powell et al.Biotechnol. Bioeng., 33:173 (1989)).

Numerous methods have been developed for the immobilization of proteinsand other biomolecules onto solid or liquid supports (see, e.g.,Mosbach, Methods in Enzymology, 44 (1976); Weetall, Immobilized Enzymes,Antigens, Antibodies, and Peptides, (1975); Kennedy et al., Solid PhaseBiochemistry, Analytical and Synthetic Aspects, Scouten, ed., pp.253–391 (1983); see, generally, Affinity Techniques. EnzymePurification: Part B. Methods in Enzymology, Vol. 34, ed. W. B. Jakoby,M. Wilchek, Acad. Press, N.Y. (1974); and Immobilized Biochemicals andAffinity Chromatography, Advances in Experimental Medicine and Biology,vol. 42, ed. R. Dunlap, Plenum Press, N.Y. (1974)).

Among the most commonly used methods are absorption and ad-sorption orcovalent binding to the support, either directly or via a linker, suchas the numerous disulfide linkages, thioether bonds, hindered disulfidebonds, and covalent bonds between free reactive groups, such as amineand thiol groups, known to those of skill in art (see, e.g., the PIERCECATALOG, ImmunoTechnology Catalog & Handbook, 1992–1993, which describesthe preparation of and use of such reagents and provides a commercialsource for such reagents; Wong, Chemistry of Protein Conjugation andCross Linking, CRC Press (1993); see also DeWitt et al., Proc. Natl.Acad. Sci. U.S.A., 90:6909 (1993); Zuckermann et al., J. Am. Chem. Soc.,114:10646 (1992); Kurth et al., J. Am. Chem. Soc., 116:2661 (1994);Ellman et al., Proc. Natl. Acad. Sci. U.S.A., 91:4708 (1994);Sucholeiki, Tetrahedron Lttrs., 35:7307 (1994); Su-Sun Wang, J. Org.Chem., 41:3258 (1976); Padwa et al., J. Org. Chem., 41:3550 (1971); andVedejs et al., J. Org. Chem., 49:575 (1984), which describephotosensitive linkers).

To effect immobilization, a composition containing the protein or otherbiomolecule is contacted with a support material such as alumina,carbon, an ion-exchange resin, cellulose, glass or a ceramic.Fluorocarbon polymers have been used as supports to which biomoleculeshave been attached by adsorption (see, U.S. Pat. No. 3,843,443;Published International PCT Application WO/86 03840).

J. Prognosis and Diagnosis

MTSP10 polypeptide proteins, domains, analogs, and derivatives thereof,and encoding nucleic acids (and sequences complementary thereto), andanti-MTSP10 polypeptide antibodies, can be used in diagnostics,particularly diagnosis of lung, head and neck, such as esophagealtumors, prostate, colon, ovary, cervix, breast and pancreas cancers.Such molecules can be used in assays, such as immunoassays, to detect,prognose, diagnose, or monitor various conditions, diseases, anddisorders affecting MTSP10 polypeptide expression, or monitor thetreatment thereof. For purposes herein, the presence of MTSP10s in bodyfluids or tumor tissues are of particular interest.

In particular, such an immunoassay is carried out by a method includingcontacting a sample derived from a patient with an anti-MTSP10polypeptide antibody under conditions such that specific binding canoccur, and detecting or measuring the amount of any specific binding bythe antibody. Such binding of antibody, in tissue sections, can be usedto detect aberrant MTSP10 polypeptide localization or aberrant (e.g.,increased, decreased or absent) levels of MTSP10 polypeptide. In aspecific embodiment, antibody to an MTSP10 polypeptide can be used toassay in a patient tissue or body fluid, such as serum, sample for thepresence of MTSP10 polypeptide where an aberrant level of MTSP10polypeptide is an indication of a diseased condition.

The immunoassays which can be used include but are not limited tocompetitive and non-competitive assay systems using techniques such aswestern blots, radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoprecipitation assays, precipitinreactions, gel diffusion precipitin reactions, immunodiffusion assays,agglutination assays, complement-fixation assays, immunoradiometricassays, fluorescent immunoassays and protein A immunoassays.

MTSP10 polypeptide genes and related nucleic acid sequences andsubsequences, including complementary sequences, also can be used inhybridization assays. MTSP10 polypeptide nucleic acid sequences, orsubsequences thereof containing about at least 8 nucleotides, generally14 or 16 or 30 or more, generally less than 1000 or up to 100,continugous nucleotides can be used as hybridization probes.Hybridization assays can be used to detect, prognose, diagnose, ormonitor conditions, disorders, or disease states associated withaberrant changes in MTSP10 polypeptide expression and/or activity asdescribed herein. In particular, such a hybridization assay is carriedout by a method by contacting a sample containing nucleic acid with anucleic acid probe capable of hybridizing to MTSP10 polypeptide encodingDNA or RNA, under conditions such that hybridization can occur, anddetecting or measuring any resulting hybridization.

In a specific embodiment, a method of diagnosing a disease or disordercharacterized by detecting an aberrant level of an MTSP10 polypeptide ina subject is provided herein by measuring the level of the DNA, RNA,protein or functional activity of the MTSP10 polypeptide in a samplederived from the subject, wherein an increase or decrease in the levelof the DNA, RNA, protein or functional activity of the MTSP10polypeptide, relative to the level of the DNA, RNA, protein orfunctional activity found in an analogous sample not having the diseaseor disorder indicates the presence of the disease or disorder in thesubject.

Kits for diagnostic use are also provided, that contain in one or morecontainers an anti-MTSP10 polypeptide antibody, and, optionally, alabeled binding partner to the antibody. Alternatively, the anti-MTSP10polypeptide antibody can be labeled (with a detectable marker, e.g., achemiluminescent, enzymatic, fluorescent, or radioactive moiety). A kitis also provided that includes in one or more containers a nucleic acidprobe capable of hybridizing to the MTSP10 polypeptide-encoding nucleicacid. In a specific embodiment, a kit can include in one or morecontainers a pair of primers (e.g., each in the size range of 6–30nucleotides) that are capable of priming amplification [e.g., bypolymerase chain reaction (see e.g., Innis et al., 1990, PCR Protocols,Academic Press, Inc., San Diego, Calif.), ligase chain reaction (see EP320,308) use of Qβ replicase, cyclic probe reaction, or other methodsknown in the art under appropriate reaction conditions of at least aportion of an MTSP10 polypeptide-encoding nucleic acid. A kit canoptionally further include in a container a predetermined amount of apurified MTSP10 polypeptide or nucleic acid, e.g., for use as a standardor control.

K. Pharmaceutical Compositions and Modes of Administration

1. Components of the Compositions

Pharmaceutical compositions containing the identified compounds thatmodulate the activity of an MTSP10 polypeptide are provided herein. Alsoprovided are combinations of a compound that modulates the activity ofan MTSP10 polypeptide and another treatment or compound for treatment ofa neoplastic disorder, such as a chemotherapeutic compound.

The MTSP10 polypeptide modulator and the anti-tumor agent can bepackaged as separate compositions for administration together orsequentially or intermittently. Alternatively, they can provided as asingle composition for administration or as two compositions foradministration as a single composition. The combinations can be packagedas kits.

a. MTSP10 Polypeptide Inhibitors

Any MTSP10 polypeptide inhibitors, including those described herein whenused alone or in combination with other compounds, that can alleviate,reduce, ameliorate, prevent, or place or maintain in a state ofremission of clinical symptoms or diagnostic markers associated withneoplastic diseases, including undesired and/or uncontrolledangiogenesis, can be used in the present combinations.

In one embodiment, the MTSP10 polypeptide inhibitor is an antibody orfragment thereof that specifically reacts with an MTSP10 polypeptide orthe protease domain thereof, an inhibitor of the MTSP10 polypeptideproduction, an inhibitor of MTSP10 polypeptide membrane-localization, orany inhibitor of the expression of or, especially, the activity of anMTSP10 polypeptide.

b. Anti-angiogenic Agents and Anti-tumor Agents

Any anti-angiogenic agents and anti-tumor agents, including thosedescribed herein, when used alone or in combination with othercompounds, that can alleviate, reduce, ameliorate, prevent, or place ormaintain in a state of remission of clinical symptoms or diagnosticmarkers associated with undesired and/or uncontrolled angiogenesisand/or tumor growth and metastasis, particularly solid neoplasms,vascular malformations and cardiovascular disorders, chronicinflammatory diseases and aberrant wound repairs, circulatory disorders,crest syndromes, dermatological disorders, or ocular disorders, can beused in the combinations. Also contemplated are anti-tumor agents foruse in combination with an inhibitor of an MTSP10 polypeptide.

c. Anti-tumor Agents and Anti-angiogenic Agents

The compounds identified by the methods provided herein or providedherein can be used in combination with anti-tumor agents and/oranti-angiogenesis agents.

2. Formulations and Route of Administration

The compounds herein and agents can be formulated as pharmaceuticalcompositions, typically for single dosage administration. Theconcentrations of the compounds in the formulations are effective fordelivery of an amount, upon administration, that is effective for theintended treatment. Typically, the compositions are formulated forsingle dosage administration. To formulate a composition, the weightfraction of a compound or mixture thereof is dissolved, suspended,dispersed or otherwise mixed in a selected vehicle at an effectiveconcentration such that the treated condition is relieved orameliorated. Pharmaceutical carriers or vehicles suitable foradministration of the compounds provided herein include any suchcarriers known to those skilled in the art to be suitable for theparticular mode of administration.

In addition, the compounds can be formulated as the solepharmaceutically active ingredient in the composition or can be combinedwith other active ingredients. Liposomal suspensions, includingtissue-targeted liposomes, can also be suitable as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art. For example, liposome formulations can beprepared as described in U.S. Pat. No. 4,522,811.

The active compound is included in the pharmaceutically acceptablecarrier in an amount sufficient to exert a therapeutically useful effectin the absence of undesirable side effects on the patient treated. Thetherapeutically effective concentration can be determined empirically bytesting the compounds in known in vitro and in vivo systems, such as theassays provided herein.

The concentration of active compound in the drug composition depends onabsorption, inactivation and excretion rates of the active compound, thephysicochemical characteristics of the compound, the dosage schedule,and amount administered as well as other factors known to those of skillin the art.

Typically a therapeutically effective dosage is contemplated. Theamounts administered can be on the order of 0.001 to 1 mg/ml, includingabout 0.005–0.05 mg/ml and about 0.01 mg/ml, of blood volume.Pharmaceutical dosage unit forms are prepared to provide from about 1 mgto about 1000 mg, including from about 10 to about 500 mg, and includingabout 25–75 mg of the essential active ingredient or a combination ofessential ingredients per dosage unit form. The precise dosage can beempirically determined.

The active ingredient can be administered at once, or can be dividedinto a number of smaller doses to be administered at intervals of time.It is understood that the precise dosage and duration of treatment is afunction of the disease being treated and can be determined empiricallyusing known testing protocols or by extrapolation from in vivo or invitro test data. It is to be noted that concentrations and dosage valuescan also vary with the severity of the condition to be alleviated. It isto be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions, and that theconcentration ranges set forth herein are exemplary only and are notintended to limit the scope or use of the claimed compositions andcombinations containing them.

Pharmaceutically acceptable derivatives include acids, salts, esters,hydrates, solvates and prodrug forms. The derivative is typicallyselected such that its pharmacokinetic properties are superior to thecorresponding neutral compound.

Thus, effective concentrations or amounts of one or more of thecompounds provided herein or pharmaceutically acceptable derivativesthereof are mixed with a suitable pharmaceutical carrier or vehicle forsystemic, topical or local administration to form pharmaceuticalcompositions. Compounds are included in an amount effective forameliorating or treating the disorder for which treatment iscontemplated. The concentration of active compound in the compositiondepends on absorption, inactivation, excretion rates of the activecompound, the dosage schedule, amount administered, particularformulation as well as other factors known to those of skill in the art.

Solutions or suspensions used for parenteral, intradermal, subcutaneous,or topical application can include any of the following components: asterile diluent, such as water for injection, saline solution, fixedoil, polyethylene glycol, glycerine, propylene glycol or other syntheticsolvent; antimicrobial agents, such as benzyl alcohol and methylparabens; antioxidants, such as ascorbic acid and sodium bisulfite;chelating agents, such as ethylenediaminetetraacetic acid (EDTA);buffers, such as acetates, citrates and phosphates; and agents for theadjustment of tonicity such as sodium chloride or dextrose. Parenteralpreparations can be enclosed in ampules, disposable syringes or singleor multiple dose vials made of glass, plastic or other suitablematerial.

In instances in which the compounds exhibit insufficient solubility,methods for solubilizing compounds can be used. Such methods are knownto those of skill in this art, and include, but are not limited to,using cosolvents, such as dimethylsulfoxide (DMSO), using surfactants,such as Tween®, or dissolution in aqueous sodium bicarbonate.Derivatives of the compounds, such as prodrugs of the compounds can alsobe used in formulating effective pharmaceutical compositions. Forophthalmic indications, the compositions are formulated in anophthalmically acceptable carrier. For the ophthalmic uses herein, localadministration, either by topical administration or by injection arecontemplated. Time release formulations are also desirable. Typically,the compositions are formulated for single dosage administration, sothat a single dose administers an effective amount.

Upon mixing or addition of the compound with the vehicle, the resultingmixture can be a solution, suspension, emulsion or other composition.The form of the resulting mixture depends upon a number of factors,including the intended mode of administration and the solubility of thecompound in the selected carrier or vehicle. If necessary,pharmaceutically acceptable salts or other derivatives of the compoundsare prepared.

The compound is included in the pharmaceutically acceptable carrier inan amount sufficient to exert a therapeutically useful effect in theabsence of undesirable side effects on the patient treated. It isunderstood that number and degree of side effects depends upon thecondition for which the compounds are administered. For example, certaintoxic and undesirable side effects are tolerated when treatinglife-threatening illnesses that would not be tolerated when treatingdisorders of lesser consequence.

The compounds also can be mixed with other active materials, that do notimpair the desired action, or with materials that supplement the desiredaction known to those of skill in the art. The formulations of thecompounds and agents for use herein include those suitable for oral,rectal, topical, inhalational, buccal (e.g., sublingual), parenteral(e.g., subcutaneous, intramuscular, intradermal, or intravenous),transdermal administration or any route. The most suitable route in anygiven case depends on the nature and severity of the condition beingtreated and on the nature of the particular active compound which isbeing used. The formulations are provided for administration to humansand animals in unit dosage forms, such as tablets, capsules, pills,powders, granules, sterile parenteral solutions or suspensions, and oralsolutions or suspensions, and oil-water emulsions containing suitablequantities of the compounds or pharmaceutically acceptable derivativesthereof. The pharmaceutically therapeutically active compounds andderivatives thereof are typically formulated and administered inunit-dosage forms or multiple-dosage forms. Unit-dose forms as usedherein refers to physically discrete units suitable for human and animalsubjects and packaged individually as is known in the art. Eachunit-dose contains a predetermined quantity of the therapeuticallyactive compound sufficient to produce the desired therapeutic effect, inassociation with the required pharmaceutical carrier, vehicle ordiluent. Examples of unit-dose forms include ampoules and syringes andindividually packaged tablets or capsules. Unit-dose forms can beadministered in fractions or multiples thereof. A multiple-dose form isa plurality of identical unit-dosage forms packaged in a singlecontainer to be administered in segregated unit-dose form. Examples ofmultiple-dose forms include vials, bottles of tablets or capsules orbottles of pints or gallons. Hence, multiple dose form is a multiple ofunit-doses which are not segregated in packaging.

The composition can contain along with the active ingredient: a diluentsuch as lactose, sucrose, dicalcium phosphate, orcarboxymethyl-cellulose; a lubricant, such as magnesium stearate,calcium stearate and talc; and a binder such as starch, natural gums,such as gum acaciagelatin, glucose, molasses, polvinylpyrrolidine,celluloses and derivatives thereof, povidone, crospovidones and othersuch binders known to those of skill in the art. Liquid pharmaceuticallyadministrable compositions can, for example, be prepared by dissolving,dispersing, or otherwise mixing an active compound as defined above andoptional pharmaceutical adjuvants in a carrier, such as, for example,water, saline, aqueous dextrose, glycerol, glycols, ethanol, and thelike, to thereby form a solution or suspension. If desired, thepharmaceutical composition to be administered can also contain minoramounts of nontoxic auxiliary substances such as wetting agents,emulsifying agents, or solubilizing agents, pH buffering agents and thelike, for example, acetate, sodium citrate, cyclodextrine derivatives,sorbitan monolaurate, triethanolamine sodium acetate, triethanolamineoleate, and other such agents. Methods of preparing such dosage formsare known, or will be apparent, to those skilled in this art (see, e.g.,Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa., 15th Edition, 1975). The composition or formulation to beadministered contains a quantity of the active compound in an amountsufficient to alleviate the symptoms of the treated subject.

Dosage forms or compositions containing active ingredient in the rangeof 0.005% to 100% with the balance made up from non-toxic carrier can beprepared. For oral administration, the pharmaceutical compositions cantake the form of, for example, tablets or capsules prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidoneor hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g., magnesium stearate, talc or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulphate). The tablets can be coated by methods well-known in theart.

The pharmaceutical preparation can also be in liquid form, for example,solutions, syrups or suspensions, or can be presented as a drug productfor reconstitution with water or other suitable vehicle before use. Suchliquid preparations can be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, or fractionated vegetable oils); andpreservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbicacid).

Formulations suitable for rectal administration can be presented as unitdose suppositories. These can be prepared by admixing the activecompound with one or more conventional solid carriers, for example,cocoa butter, and then shaping the resulting mixture.

Formulations suitable for topical application to the skin or to the eyegenerally are formulated as an ointment, cream, lotion, paste, gel,spray, aerosol and oil. Carriers which can be used include vaseline,lanoline, polyethylene glycols, alcohols, and combinations of two ormore thereof. The topical formulations can further advantageouslycontain 0.05 to 15 percent by weight of thickeners selected from amonghydroxypropyl methyl cellulose, methyl cellulose, polyvinylpyrrolidone,polyvinyl alcohol, poly (alkylene glycols), poly/hydroxyalkyl,(meth)acrylates or poly(meth)acrylamides. A topical formulation is oftenapplied by instillation or as an ointment into the conjunctival sac. Italso can be used for irrigation or lubrication of the eye, facialsinuses, and external auditory meatus. It can also be injected into theanterior eye chamber and other places. The topical formulations in theliquid state can be also present in a hydrophilic three-dimensionalpolymer matrix in the form of a strip, contact lens, and the like fromwhich the active components are released.

For administration by inhalation, the compounds for use herein can bedelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebulizer, with the use of a suitable propellant, e.g.,dichlorodi-fluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin, for use in an inhaler or insufflator can beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

Formulations suitable for buccal (sublingual) administration include,for example, lozenges containing the active compound in a flavored base,usually sucrose and acacia or tragacanth; and pastilles containing thecompound in an inert base such as gelatin and glycerin or sucrose andacacia.

The compounds can be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection can be presented in unit dosage form, e.g., in ampules orin multi-dose containers, with an added preservative. The compositionscan be suspensions, solutions or emulsions in oily or aqueous vehicles,and can contain formulatory agents such as suspending, stabilizingand/or dispersing agents. Alternatively, the active ingredient can be inpowder form for reconstitution with a suitable vehicle, e.g., sterilepyrogen-free water or other solvents, before use.

Formulations suitable for transdermal administration can be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Such patchessuitably contain the active compound as an optionally buffered aqueoussolution of, for example, 0.1 to 0.2 M concentration with respect to theactive compound. Formulations suitable for transdermal administrationcan also be delivered by iontophoresis (see, e.g., PharmaceuticalResearch 3 (6), 318 (1 986)) and typically take the form of anoptionally buffered aqueous solution of the active compound.

The pharmaceutical compositions can also be administered by controlledrelease means and/or delivery devices (see, e.g., in U.S. Pat. Nos.3,536,809; 3,598,123; 3,630,200; 3,845,770; 3,847,770; 3,916,899;4,008,719; 4,687,610; 4,769,027; 5,059,595; 5,073,543; 5,120,548;5,354,566; 5,591,767; 5,639,476; 5,674,533 and 5,733,566).

Desirable blood levels can be maintained by a continuous infusion of theactive agent as ascertained by plasma levels. It should be noted thatthe attending physician would know how to and when to terminate,interrupt or adjust therapy to lower dosage due to toxicity, or bonemarrow, liver or kidney dysfunctions. Conversely, the attendingphysician would also know how to and when to adjust treatment to higherlevels if the clinical response is not adequate (precluding toxic sideeffects).

The efficacy and/or toxicity of the MTSP10 polypeptide inhibitor(s),alone or in combination with other agents also can be assessed by themethods known in the art (See generally, O'Reilly, Investigational NewDrugs, 15:5–13 (1997)).

The active compounds or pharmaceutically acceptable derivatives can beprepared with carriers that protect the compound against rapidelimination from the body, such as time release formulations orcoatings.

Kits containing the compositions and/or the combinations withinstructions for administration thereof are provided. The kit canfurther include a needle or syringe, typically packaged in sterile form,for injecting the complex, and/or a packaged alcohol pad. Instructionsare optionally included for administration of the active agent by aclinician or by the patient.

Finally, the compounds or MTSP10 polypeptides or protease domainsthereof or compositions containing any of the preceding agents can bepackaged as articles of manufacture containing packaging material, acompound or suitable derivative thereof provided herein, which iseffective for treatment of a diseases or disorders contemplated herein,within the packaging material, and a label that indicates that thecompound or a suitable derivative thereof is for treating the diseasesor disorders contemplated herein. The label can optionally include thedisorders for which the therapy is warranted.

L. Methods of Treatment

The compounds identified by the methods herein are used for treating orpreventing neoplastic diseases in an animal, particularly a mammal,including a human, is provided herein. In one embodiment, the methodincludes administering to a mammal an effective amount of an inhibitorof an MTSP10 polypeptide, whereby the disease or disorder is treated orprevented.

In an embodiment, the MTSP10 polypeptide inhibitor used in the treatmentor prevention is administered with a pharmaceutically acceptable carrieror excipient. The mammal treated can be a human. The inhibitors providedherein are those identified by the screening assays. In addition,antibodies and antisense nucleic acids or double-stranded RNA (dsRNA),such as RNAi, are contemplated.

The treatment or prevention method can further include administering ananti-angiogenic treatment or agent or anti-tumor agent simultaneouslywith, prior to or subsequent to the MTSP10 polypeptide inhibitor, whichcan be any compound identified that inhibits the activity of an MTSP10polypeptide. Such compounds include small molecule modulators, anantibody or a fragment or derivative thereof containing a binding regionthereof against the MTSP10 polypeptide, an antisense nucleic acid ordouble-stranded RNA (dsRNA), such as RNAi, encoding an a portion of theMTSP10 polypeptide or complementary to thereto, and a nucleic acidcontaining at least a portion of a gene encoding the MTSP10 polypeptideinto which a heterologous nucleotide sequence has been inserted suchthat the heterologous sequence inactivates the biological activity of atleast a portion of the gene encoding the MTSP10 polypeptide, in whichthe portion of the gene encoding the MTSP10 polypeptide flanks theheterologous sequence to promote homologous recombination with a genomicgene encoding the MTSP10 polypeptide. In addition, such molecules aregenerally less than about 1000 nt long.

1. Antisense Treatment

In a specific embodiment, as described hereinabove, MTSP10 polypeptidefunction is reduced or inhibited by MTSP10 polypeptide antisense nucleicacids, to treat or prevent neoplastic disease. The therapeutic orprophylactic use of nucleic acids of at least six nucleotides, generallyup to about 150 nucleotides, that are antisense to a gene or cDNAencoding MTSP10 polypeptide or a portion thereof is provided. An MTSP10polypeptide “antisense” nucleic acid as used herein refers to a nucleicacid capable of hybridizing to a portion of an MTSP10 polypeptide RNA(generally mRNA) by virtue of some sequence complementarity, andgenerally under high stringency conditions. The antisense nucleic acidcan be complementary to a coding and/or noncoding region of an MTSP10polypeptide mRNA. Such antisense nucleic acids have utility astherapeutics that reduce or inhibit MTSP10 polypeptide function, and canbe used in the treatment or prevention of disorders as described supra.

The MTSP10 polypeptide antisense nucleic acids are of at least sixnucleotides and are generally oligonucleotides (ranging from 6 to about150 nucleotides including 6 to 50 nucleotides). The antisense moleculecan be complementary to all or a portion of the protease domain. Forexample, the oligonucleotide is at least 10 nucleotides, at least 15nucleotides, at least 100 nucleotides, or at least 125 nucleotides. Theoligonucleotides can be DNA or RNA or chimeric mixtures or derivativesor modified versions thereof, single-stranded or double-stranded. Theoligonucleotide can be modified at the base moiety, sugar moiety, orphosphate backbone. The oligonucleotide can include other appendinggroups such as peptides, or agents facilitating transport across thecell membrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci.U.S.A. 86:6553–6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci.U.S.A. 84:648–652 (1987); PCT Publication No. WO 88/09810, publishedDec. 15, 1988) or blood-brain barrier (see, e.g., PCT Publication No. WO89/10134, published Apr. 25, 1988), hybridization-triggered cleavageagents (see, e.g., Krol et al., BioTechniques 6:958–976 (1988)) orintercalating agents (see, e.g., Zon, Pharm. Res. 5:539–549 (1988)).

The MTSP10 polypeptide antisense nucleic acid generally is anoligonucleotide, typically single-stranded DNA or RNA or an analogthereof or mixtures thereof. For example, the oligonucleotide includes asequence antisense to a portion of a nucleic acid that encodes a humanMTSP10 polypeptide. The oligonucleotide can be modified at any positionon its structure with substituents generally known in the art.

The MTSP10 polypeptide antisense oligonucleotide can include at leastone modified base moiety which is selected from the group including, butnot limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

In another embodiment, the oligonucleotide includes at least onemodified sugar moiety selected from the group including but not limitedto arabinose, 2-fluoroarabinose, xylulose, and hexose. Theoligonucleotide can include at least one modified phosphate backboneselected from a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

The oligonucleotide can be an α-anomeric oligonucleotide. An α-anomericoligonucleotide forms specific double-stranded hybrids withcomplementary RNA in which the strands run parallel to each other(Gautier et al., Nucl. Acids Res. 15:6625–6641 (1987)).

The oligonucleotide can be conjugated to another molecule, such as, butare not limited to, a peptide, hybridization triggered cross-linkingagent, transport agent or a hybridization-triggered cleavage agent. Theoligonucleotides can be synthesized by standard methods known in theart, e.g. by use of an automated DNA synthesizer (such as arecommercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides can be synthesized by themethod of Stein et al. (Nucl. Acids Res. 16:3209 (1988)),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A.85:7448–7451 (1988)), etc.

In a specific embodiment, the MTSP10 polypeptide antisenseoligonucleotide includes catalytic RNA or a ribozyme (see, e.g., PCTInternational Publication WO 90/11364, published Oct. 4, 1990; Sarver etal., Science 247:1222–1225 (1990)). In another embodiment, theoligonucleotide is a 2′-O-methylribonucleotide (Inoue et al., Nucl.Acids Res. 15:6131–6148 (1987)), or a chimeric RNA-DNA analogue (Inoueet al., FEBS Lett. 215:327–330 (1987)). Alternatively, theoligonucleotide can be double-stranded RNA (dsRNA) such as RNAi.

In an alternative embodiment, the MTSP10 polypeptide antisense nucleicacid is produced intracellularly by transcription from an exogenoussequence. For example, a vector can be introduced in vivo such that itis taken up by a cell, within which cell the vector or a portion thereofis transcribed, producing an antisense nucleic acid (RNA). Such a vectorwould contain a sequence encoding the MTSP10 polypeptide antisensenucleic acid. Such a vector can remain episomal or become chromosomallyintegrated, as long as it can be transcribed to produce the desiredantisense RNA. Such vectors can be constructed by recombinant DNAtechnology methods standard in the art. Vectors can be plasmid, viral,or others known in the art, used for replication and expression inmammalian cells. Expression of the sequence encoding the MTSP10polypeptide antisense RNA can be by any promoter known in the art to actin mammalian, including human, cells. Such promoters can be inducible orconstitutive. Such promoters include but are not limited to: the SV40early promoter region (Bernoist and Chambon, Nature 290:304–310 (1981),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., Cell 22:787–797 (1980), the herpes thymidinekinase promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A.78:1441–1445 (1981), the regulatory sequences of the metallothioneingene (Brinster et al., Nature 296:39–42 (1982), etc.

The antisense nucleic acids include sequence complementary to at least aportion of an RNA transcript of an MTSP10 polypeptide gene, including ahuman MTSP10 polypeptide gene. Absolute complementarily is not required.

The amount of MTSP10 polypeptide antisense nucleic acid that iseffective in the treatment or prevention of neoplastic disease dependson the nature of the disease, and can be determined empirically bystandard clinical techniques. Where possible, it is desirable todetermine the antisense cytotoxicity in cells in vitro, and then inuseful animal model systems prior to testing and use in humans.

2. RNA Interference

RNA interference (RNAi) (see, e.g. Chuang et al. (2000) Proc. Natl.Acad. Sci. U.S.A. 97:4985) can be employed to inhibit the expression ofa gene encoding an MTSP10. Interfering RNA (RNAi) fragments,particularly double-stranded (ds) RNAi, can be used to generateloss-of-MTSP10 function. Methods relating to the use of RNAi to silencegenes in organisms including, mammals, C. elegans, Drosophila andplants, and humans are known (see, e.g., Fire et al. (1998) Nature391:806–811 Fire (1999) Trends Genet. 15:358–363; Sharp (2001) GenesDev. 15:485–490; Hammond, et al. (2001) Nature Rev. Genet. 2:110–1119;Tuschl (2001) Chem. Biochem. 2:239–245; Hamilton et al. (1999) Science286:950–952; Hammond et al. (2000) Nature 404:293–296; Zamore et al.(2000) Cell 101:25–33; Bernstein et al. (2001) Nature 409: 363–366;Elbashir et al. (2001) Genes Dev. 15:188–200; Elbashir et al. (2001)Nature 411:494–498; International PCT application No. WO 01/29058;International PCT application No. WO 99/32619).

Double-stranded RNA (dsRNA)-expressing constructs are introduced into ahost, such as an animal or plant using, a replicable vector that remainsepisomal or integrates into the genome. By selecting appropriatesequences, expression of dsRNA can interfere with accumulation ofendogenous mRNA encoding an MTSP10. RNAi also can be used to inhibitexpression in vitro. Regions include at least about 21 (or 21)nucleotides that are selective (i.e. unique) for MTSP10 are used toprepare the RNAi. Smaller fragments of about 21 nucleotides can betransformed directly (i.e., in vitro or in vivo) into cells; larger RNAidsRNA molecules are generally introduced using vectors that encode them.dsRNA molecules are at least about 21 bp long or longer, such as 50,100, 150, 200 and longer. Methods, reagents and protocols forintroducing nucleic acid molecules in to cells in vitro and in vivo areknown to those of skill in the art.

3. Gene Therapy

In an exemplary embodiment, nucleic acids that include a sequence ofnucleotides encoding an MTSP10 polypeptide or functional domains orderivative thereof, are administered to promote MTSP10 polypeptidefunction, by way of gene therapy. Gene therapy refers to therapyperformed by the administration of a nucleic acid to a subject. In thisembodiment, the nucleic acid produces its encoded protein that mediatesa therapeutic effect by promoting MTSP10 polypeptide function. Any ofthe methods for gene therapy available in the art can be used (see,Goldspiel et al., Clinical Pharmacy 12:488–505 (1993); Wu and Wu,Biotherapy 3:87–95 (1991); Tolstoshev, An. Rev. Pharmacol. Toxicol.32:573–596 (1993); Mulligan, Science 260:926–932 (1993); and Morgan andAnderson, An. Rev. Biochem. 62:191–217 (1993); TIBTECH 11 (5):155–215(1993).

For example, one therapeutic composition for gene therapy includes anMTSP10 polypeptide-encoding nucleic acid that is part of an expressionvector that expresses an MTSP10 polypeptide or domain, fragment orchimeric protein thereof in a suitable host. In particular, such anucleic acid has a promoter operably linked to the MTSP10 polypeptidecoding region, the promoter being inducible or constitutive, and,optionally, tissue-specific. In another particular embodiment, a nucleicacid molecule is used in which the MTSP10 polypeptide coding sequencesand any other desired sequences are flanked by regions that promotehomologous recombination at a desired site in the genome, thus providingfor intrachromosomal expression of the SP protein nucleic acid (Kollerand Smithies, Proc. Natl. Acad. Sci. USA 86:8932–8935 (1989); Zijlstraet al., Nature 342:435–438 (1989)).

Delivery of the nucleic acid into a patient can be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vector, or indirect, in which case, cells arefirst transformed with the nucleic acid in vitro, then transplanted intothe patient. These two approaches are known, respectively, as in vivo orex vivo gene therapy.

In a specific embodiment, the nucleic acid is directly administered invivo, where it is expressed to produce the encoded product. This can beaccomplished by any of numerous methods known in the art, e.g., byconstructing it as part of an appropriate nucleic acid expression vectorand administering it so that it becomes intracellular, e.g., byinfection using a defective or attenuated retroviral or other viralvector (see U.S. Pat. No. 4,980,286), or by direct injection of nakedDNA, or by use of microparticle bombardment (e.g., a gene gun;Biolistic, Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, encapsulation in liposomes, microparticles, ormicrocapsules, or by administering it in linkage to a peptide which isknown to enter the nucleus, by administering it in linkage to a ligandsubject to receptor-mediated endocytosis (see e.g., Wu and Wu, J. Biol.Chem. 262:4429–4432 (1987)) (which can be used to target cell typesspecificall expressing the receptors), etc. In another embodiment, anucleic acid-ligand complex can be formed in which the ligand is afusogenic viral peptide to disrupt endosomes, allowing the nucleic acidto avoid lysosomal degradation. In yet another embodiment, the nucleicacid can be targeted in vivo for cell specific uptake and expression, bytargeting a specific receptor (see, e.g., PCT Publications WO 92/06180dated Apr. 16, 1992 (Wu et al.); WO 92/22635 dated Dec. 23, 1992 (Wilsonet al.); WO92/20316 dated Nov. 26, 1992 (Findeis et al.); WO93/14188dated Jul. 22, 1993 (Clarke et al.), WO 93/20221 dated Oct. 14, 1993(Young)). Alternatively, the nucleic acid can be introducedintracellularly and incorporated within host cell DNA for expression, byhomologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci.USA 86:8932–8935 (1989); Zijlstra et al., Nature 342:435–438 (1989)).

In a specific embodiment, a viral vector that contains the MTSP10polypeptide nucleic acid is used. For example, a retroviral vector canbe used (see Miller et al., Meth. Enzymol. 217:581–599 (1993)). Theseretroviral vectors have been modified to delete retroviral sequencesthat are not necessary for packaging of the viral genome and integrationinto host cell DNA. The MTSP10 polypeptide nucleic acid to be used ingene therapy is cloned into the vector, which facilitates delivery ofthe gene into a patient. More detail about retroviral vectors can befound in Boesen et al., Biotherapy 6:291–302 (1994), which describes theuse of a retroviral vector to deliver the mdr1 gene to hematopoieticstem cells in order to make the stem cells more resistant tochemotherapy. Other references illustrating the use of retroviralvectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644–651(1994); Kiem et al., Blood 83:1467–1473 (1994); Salmons and Gunzberg,Human Gene Therapy 4:129–141 (1993); and Grossman and Wilson, Curr.Opin. in Genetics and Devel. 3:110–114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499–503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3–10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431–434 (1991); Rosenfeld et al., Cell 68:143–155 (1992);and Mastrangeli et al., J. Clin. Invest. 91:225–234 (1993).

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289–300 (1993).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see e.g., Loeffler and Behr, Meth. Enzymol.217:599–618 (1993); Cohen et al., Meth. Enzymol. 217:618–644 (1993);Cline, Pharmac. Ther. 29:69–92 (1985)) and can be used, provided thatthe necessary developmental and physiological functions of the recipientcells are not disrupted. The technique should provide for the stabletransfer of the nucleic acid to the cell, so that the nucleic acid isexpressible by the cell and generally heritable and expressible by itscell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. In an embodiment, epithelial cells areinjected, e.g., subcutaneously. In another embodiment, recombinant skincells can be applied as a skin graft onto the patient. Recombinant bloodcells (e.g., hematopoietic stem or progenitor cells) can be administeredintravenously. The amount of cells envisioned for use depends on thedesired effect, patient state, etc., and can be determined by oneskilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., suchas stem cells obtained from bone marrow, umbilical cord blood,peripheral blood, fetal liver, and other sources thereof.

For example, a cell used for gene therapy is autologous to the patient.In an embodiment in which recombinant cells are used in gene therapy, anMTSP10 polypeptide nucleic acid is introduced into the cells such thatit is expressible by the cells or their progeny, and the recombinantcells are then administered in vivo for therapeutic effect. In aspecific embodiment, stem or progenitor cells are used. Any stem and/orprogenitor cells which can be isolated and maintained in vitro canpotentially be used in accordance with this embodiment. Such stem cellsinclude but are not limited to hematopoietic stem cells (HSC), stemcells of epithelial tissues such as the skin and the lining of the gut,embryonic heart muscle cells, liver stem cells (PCT Publication WO94/08598, dated Apr. 28, 1994), and neural stem cells (Stemple andAnderson, Cell 71:973–985 (1992)).

Epithelial stem cells (ESCs) or keratinocytes can be obtained fromtissues such as the skin and the lining of the gut by known procedures(Rheinwald, Meth. Cell Bio. 21A:229 (1980)). In stratified epithelialtissue such as the skin, renewal occurs by mitosis of stem cells withinthe germinal layer, the layer closest to the basal lamina. Stem cellswithin the lining of the gut provide for a rapid renewal rate of thistissue. ESCs or keratinocytes obtained from the skin or lining of thegut of a patient or donor can be grown in tissue culture (Rheinwald,Meth. Cell Bio. 21A:229 (1980); Pittelkow and Scott, Cano Clinic Proc.61:771 (1986)). If the ESCs are provided by a donor, a method forsuppression of host versus graft reactivity (e.g., irradiation, drug orantibody administration to promote moderate immunosuppression) also canbe used.

With respect to hematopoietic stem cells (HSC), any technique whichprovides for the isolation, propagation, and maintenance in vitro of HSCcan be used in this embodiment. Techniques by which this can beaccomplished include (a) the isolation and establishment of HSC culturesfrom bone marrow cells isolated from the future host, or a donor, or (b)the use of previously established long-term HSC cultures, which can beallogeneic or xenogeneic. Non-autologous HSC generally are used with amethod of suppressing transplantation immune reactions of the futurehost/patient. In a particular embodiment, human bone marrow cells can beobtained from the posterior iliac crest by needle aspiration (see, e.g.,Kodo et al., J. Clin. Invest. 73:1377–1384 (1984)). For example, theHSCs can be made highly enriched or in substantially pure form. Thisenrichment can be accomplished before, during, or after long-termculturing, and can be done by any techniques known in the art. Long-termcultures of bone marrow cells can be established and maintained byusing, for example, modified Dexter cell culture techniques (Dexter etal., J. Cell Physiol. 91:335 (1977) or Witlock-Witte culture techniques(Witlock and Witte, Proc. Natl. Acad. Sci. USA 79:3608–3612 (1982)).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy includes an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

3. Prodrugs

A method for treating tumors is provided. The method is practiced byadministering a prodrug that is cleaved at a specific site by an MTSP10to release an active drug or precursor that can be converted to activedrug in vivo. Upon contact with a cell that expresses MTSP10 activity,the prodrug is converted into an active drug. The prodrug can be aconjugate that contains the active agent, such as an anti-tumor drug,such as a cytotoxic agent, or other therapeutic agent (TA), linked to asubstrate for the targeted MTSP10, such that the drug or agent isinactive or unable to enter a cell, in the conjugate, but is activatedupon cleavage. The prodrug, for example, can contain an oligopeptide,typically a relatively short, less than about 10 amino acids peptide,that is proteolytically cleaved by the targeted MTSP10. Cytotoxicagents, include, but are not limited to, alkylating agents,antiproliferative agents and tubulin binding agents. Others include,vinca drugs, mitomycins, bleomycins and taxanes.

M. Animal Models

Transgenic animal models and animals, such as rodents, including miceand rats, cows, chickens, pigs, goats, sheep, monkeys, includinggorillas, and other primates, are provided herein. In particular,transgenic non-human animals that contain heterologous nucleic acidencoding an MTSP10 polypeptide or a transgenic animal in whichexpression of the polypeptide has been altered, such as by replacing ormodifying the promoter region or other regulatory region of theendogenous gene are provided. Such an animal can by produced bypromoting recombination between endogenous nucleic acid and an exogenousMTSP10 gene that could be over-expressed or mis-expressed, such as byexpression under a strong promoter, via homologous or otherrecombination event.

Transgenic animals can be produced by introducing the nucleic acid usingany know method of delivery, including, but not limited to,microinjection, lipofection and other modes of gene delivery into agermline cell or somatic cells, such as an embryonic stem cell.Typically the nucleic acid is introduced into a cell, such as anembryonic stem cell (ES), followed by injecting the ES cells into ablastocyst, and implanting the blastocyst into a foster mother, which isfollowed by the birth of a transgenic animal. Generally, introduction ofa heterologous nucleic acid molecule into a chromosome of the animaloccurs by a recombination between the heterologous MTSP10-encodingnucleic acid and endogenous nucleic acid. The heterologous nucleic acidcan be targeted to a specific chromosome.

In some instances, knockout animals can be produced. Such an animal canbe initially produced by promoting homologous recombination between anMTSP10 polypeptide gene in its chromosome and an exogenous MTSP10polypeptide gene that has been rendered biologically inactive (typicallyby insertion of a heterologous sequence, e.g., an antibiotic resistancegene). In one embodiment, this homologous recombination is performed bytransforming embryo-derived stem (ES) cells with a vector containing theinsertionally inactivated MTSP10 polypeptide gene, such that homologousrecombination occurs, followed by injecting the ES cells into ablastocyst, and implanting the blastocyst into a foster mother, followedby the birth of the chimeric animal (“knockout animal”) in which anMTSP10 polypeptide gene has been inactivated (see Capecchi, Science244:1288–1292 (1989)). The chimeric animal can be bred to producehomozygous knockout animals, which can then be used to produceadditional knockout animals. Knockout animals include, but are notlimited to, mice, hamsters, sheep, pigs, cattle, and other non-humanmammals. For example, a knockout mouse is produced. The resultinganimals can serve as models of specific diseases, such as cancers, thatexhibit under-expression of an MTSP10 polypeptide. Such knockout animalscan be used as animal models of such diseases e.g., to screen for ortest molecules for the ability to treat or prevent such diseases ordisorders.

Other types of transgenic animals also can be produced, including thosethat over-express the MTSP10 polypeptide. Such animals include“knock-in” animals that are animals in which the normal gene is replacedby a variant, such a mutant, an over-expressed form, or other form. Forexample, one species′, such as a rodent's endogenous gene can bereplaced by the gene from another species, such as from a human. Animalsalso can be produced by non-homologous recombination into other sites ina chromosome; including animals that have a plurality of integrationevents.

After production of the first generation transgenic animal, a chimericanimal can be bred to produce additional animals with over-expressed ormis-expressed MTSP10 polypeptides. Such animals include, but are notlimited to, mice, hamsters, sheep, pigs, cattle and other non-humanmammals. The resulting animals can serve as models of specific diseases,such as cancers, that are exhibit over-expression or mis-expression ofan MTSP10 polypeptide. Such animals can be used as animal models of suchdiseases e.g., to screen for or test molecules for the ability to treator prevent such diseases or disorders. In a specific embodiment, a mousewith over-expressed or mis-expressed MTSP10 polypeptide is produced.

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

EXAMPLE 1 Identification of MTSP10

The protein sequence of the protease domain of matriptase (MTSP1;accession number AF118224) was used to search the human HTGS (HighThroughput Genomic Sequence) database using the tblastn search andalignment algorithm, which compares a protein query sequence against anucleotide sequence database dynamically translated in all six readingframes (both strands). Several potential serine proteases wereidentified, including the one designated herein as MTSP10.

Based on the incomplete and unordered human genome sequences, MTSP10appears to be localized in either chromosome 3 (AC024887 and AC 073522clones) or chromosome 8 (AC024964 clone). A search of sequencesdeposited in GenBank and human EST database showed that no identicalsequence has been deposited.

Cloning of cDNA Encoding the Protease Domain of MTSP10

Using the nucleotide sequence of MTSP10 derived from the genomicsequence, two gene specific oligonucleotide primers were designed. Thesequence for the 5′-end primer (C8-N1A-1) was5′-CGCATCATCGGAGGCACAGACACCCT-3′ (SEQ ID No. 7) and that of the 3′-endprimer (C8-NSP1–3AS) was 5′-CCAGGGAACAAAGTTTGACACCCTTGTG -3′ (SEQ ID No.8)

A band of 690 bp was amplified from human pancreas Marathon cDNA(Clontech). Subsequent sequence analysis showed that the nucleotidesequence of this DNA fragment matched that of the genomic MTSP10 exonsequences and includes most of the MTSP10 protease domain. A stop codonwas not found in this cDNA sequence.

To obtain the 3′-end the encoding DNA of the MTSP10 protease domain,3′-RACE (rapid amplification of cNDA ends) reactions were performed onMarathon-Ready cDNA library from human pancreas (Clontech).Marathon-Ready cDNA are specifically made for RACE reactions.

The first RACE reactions were performed by PCR using Marathon cDNAadaptor primer 1 (AP1) with gene specific primers, C8-N1A-1,5′-CGCATCATCGGAGGCACAGACACCCT-3′ (SEQ ID No. 9). The PCR products werepurified from agarose gel.

A second nested PCR was then performed using Marathon cDNA adaptorprimer 2 (AP2) with gene specific primer (Ch8-NSP1–4)5′-CTCCCACTGGTCAGAGAGTTCGCAGTG-3′ (SEQ ID No. 10; using first 3′-RACEproduct as template). PCR products from RACE reactions which were largerthan 300 bp were cut out and purified from agarose gel and subclonedinto pCR2.1-TOPO cloning vector (Invitrogen, Carlsbad, Calif.). Colonyhybridization was then performed to identify positive coloniescontaining MTSP10 sequence. Positive clones were identified by colonyhybridization using a 690 bp DNA fragment containing MTSP10 proteasedomain sequence (obtained from PCR reaction with primers C8-N1A-1 andC8-NSP1–3AS) and by DNA sequencing. Sequence analysis of the 3′-RACEproducts indicated that an additional 36 bp sequence including thepresumed stop codon was obtained.

Gene Expression Profile of MTSP10 in Normal and Tumor Tissues

To obtain information regarding the gene expression profile of theMTSP10 transcript, PCR analysis was carried out on cDNA panels made fromseveral human adult tissues (Clontech, Cat. #K1420–1) cDNA panel usingMTSP10-specific primers , C8-N1A-1 (5′-CGCATCATCGGAGGCACAGACACCCT-3′;SEQ ID No. 11) and C8-N1A-2AS (5′-CTCCCCACTGCGAACTCTCTGACCAGTG-3′; SEQID No. 12). MTSP10 transcript was detected in pancreas, lung and kidney.MTSP10 transcript was also detected in small intestine Marathon-ReadycDNA (Clontech). PCR of the MTSP10 transcript from cDNA libraries madefrom several human primary tumors xenografted in nude mice (human tumormultiple tissue cDNA panel, catalog number K1522-1, CLONTECH) was alsoperformed. The MTSP10 transcript was detected in breast carcinoma(GI-101), lung carcinoma (LX-1 and GI-117), ovarian carcinoma (GI-102),and pancreatic adenocarcinoma (GI-103). The MTSP10 transcript can beweakly detected in prostatic adenocarcinoma (PC3). No apparent signalwas detected in two forms of colon adenocarcinomas (GI-112 and CX-1).The MTSP10 transcript was also detected in a CWR22R prostate tumor grownon nude mice.

PCR Amplification of cDNA Encoding Full-length Protease Domain of MTSP10

To obtain the cDNA fragment encoding the protease domain of MTSP10, anend-to-end PCR amplification using gene-specific primers and the cDNAlibrary from human pancreas was used. The two primers used were:

-   5′-CGCATCATCGGAGGCACAGACACCCT-3′ (SEQ ID No. 11) for the 5′ end; and    5′-TTACAAAAGAGAAGGGACATATTTATGAATC-3′ (SEQ ID No. 21) for the 3′    end. The sequences for both primers were derived from the cDNA    sequence of MTSP10. The 5′ primer contains the sequence that encodes    a region immediately upstream of the start of the MTSP10 protease    domain (RIIGGTDTL). The 3′ primer corresponds to the sequence    immediately after the presumed stop codon. A 720-bp fragment was    amplified from the human pancreas cDNA library. The PCR product was    isolated and purified using the QlAquick gel extraction kit (Qiagen,    Valencia, Calif.; catalog no. 28704), and was confirmed by DNA    sequencing analysis.    Serine Protease Domain of MTSP10 and Homology to Other Proteases

Sequence analysis of the translated MTSP10 protease domain sequenceindicated that MTSP10 contains a trypsin-like serine protease domaincharacterized by the presence of a protease activation cleavage site atthe beginning of the domain and the catalytic triad residues (histidine,aspartate and serine) in 3 highly-conserved regions. Alignment of theprotein sequence with that of Matriptase (accession number AF118224; seeSEQ ID Nos. 1 and 3) shows 47% identity in the protease domain.

Sequence Analysis

MTSP10 nucleic acid and protein sequences were analyzed using DNAStrider (version 1.2). The cDNA encoding the protease domain of MTSP10is 714 bp in length, which translates into a 238-amino acid protein. ThecDNA sequence for the protease domain and the translated proteinsequence of MTSP10 is as follows (see, also SEQ ID Nos. 5 and 22 and 6and 23):

Nucleotide and Amino Acid sequences of Human MTSP10 Protease Domain, anddomain organization

cDNA/Protein Sequences

Sequence Range: 1 to 2130

    10       20       30       40       50       60ATAAACCTGGTTTATACAACATCTGCCTTCTCCAAATTTTATGAGCAGTCTGTTGTTGCATATTTGGACCAAATATGTTGTAGACGGAAGAGGTTTAAAATACTCGTCAGACAACAACGT I  N  L  V  Y  T  T  S  A  F  S  K  F  Y  E  Q  S  V  V  A        70        80        90       100       110       120GATGTCAGCAGCAACAACAAAGGCGGCCTCCTTGTCCACTTTTGGATTGTTTTTGTCATGCTACAGTCGTCGTTGTTGTTTCCGCCGGAGGAACAGGTGAAAACCTAACAAAAACAGTAC D  V  S  S  N  N  K  G  G  L  L  V  H  F  W  I  V  F  V  M       130       140       150       160       170       180CCACGTGCCAAAGGCCACATCTTCTGTGAAGACTGTGTTGCCGCCATCTTGAAGGACTCCGGTGCACGGTTTCCGGTGTAGAAGACACTTCTGACACAACGGCGGTAGAACTTCCTGAGG P  R  A  K  G  H  I  F  C  E  D  C  V  A  A  I  L  K  D  S       190       200       210       220       230       240ATCCAGACAAGCATCATAAACCGGACCTCTGTGGGGAGCTTGCAGGGACTGGCTGTGGACTAGGTCTGTTCGTAGTATTTGGCCTGGAGACACCCCTCGAACGTCCCTGACCGACACCTG I  Q  T  S  I  I  N  R  T  S  V  G  S  L  Q  G  L  A  V  D       250       260       270       280       290       300ATGGACTCTGTGGTACTAAATGCTGGGCTTCGGTCAGATTACTCGTCAACCATAGGATCTTACCTGAGACACCATGATTTACGACCCGAAGCCAGTCTAATGAGCAGTTGGTATCCTAGA M  D  S  V  V  L  N  A  G  L  R  S  D  Y  S  S  T  I  G  S       310       320       330       340       350       360GACAAAGGCTGCTCTCAGTACTTCTATGCAGAGCATCTGTCTCTCCACTACCCGCTGGAGCTGTTTCCGACGAGAGTCATGAAGATACGTCTCGTAGACAGAGAGGTGATGGGCGACCTC D  K  G  C  S  Q  Y  F  Y  A  E  H  L  S  L  H  Y  P  L  E       370       380       390       400       410       420ATTTCTGCAGCCTCAGGGAGGCTGATGTGTCACTTCAAGCTGGTGGCCATAGTGGGCTACTAAAGACGTCGGAGTCCCTCCGACTACACAGTGAAGTTCGACCACCGGTATCACCCGATG I  S  A  A  S  G  R  L  M  C  H  F  K  L  V  A  I  V  G  Y       430       440       450       460       470       480CTGATTCGTCTCTCAATCAAGTCCATCCAAATCGAAGCCGACAACTGTGTCACTGACTCCGACTAAGCAGAGAGTTAGTTCAGGTAGGTTTAGCTTCGGCTGTTGACACAGTGACTGAGG L  I  R  L  S  I  K  S  I  Q  I  E  A  D  N  C  V  T  D  S       490       500       510       520       530       540CTGACCATTTACGACTCCCTTTTGCCCATCCGGAGCAGCATCTTGTACAGAATTTGTGAAGACTGGTAAATGCTGAGGGAAAACGGGTAGGCCTCGTCGTAGAACATGTCTTAAACACTT L  T  I  Y  D  S  L  L  P  I  R  S  S  I  L  Y  R  I  C  E       550       560       570       580       590       600CCCACAAGAACATTAATGTCATTTGTTTCTACAAATAATCTCATGTTGGTGACATTTAAGGGGTGTTCTTGTAATTACAGTAAACAAAGATGTTTATTAGAGTACAACCACTGTAAATTC P  T  R  T  L  M  S  F  V  S  T  N  N  L  M  L  V  T  F  K       610       620       630       640       650       660TCTCCTCATATACGGAGGCTCTCAGGAATCCGGGCATATTTTGAGGTCATTCCAGAACAAAGAGGAGTATATGCCTCCGAGAGTCCTTAGGCCCGTATAAAACTCCAGTAAGGTCTTGTT S  P  H  I  R  R  L  S  G  I  R  A  Y  F  E  V  I  P  E  Q       670       680       690       700       710       720AAGTGTGAAAACACAGTGTTGGTCAAAGACATCACTGGCTTTGAAGGGAAAATTTCAAGCTTCACACTTTTGTGTCACAACCAGTTTCTGTAGTGACCGAAACTTCCCTTTTAAAGTTCG K  C  E  N  T  V  L  V  K  D  I  T  G  F  E  G  K  I  S  S       730       740       750       760       770       780CCATATTACCCGAGCTACTATCCTCCAAAATGCAAGTGTACCTGGAAATTTCAGACTTCTGGTATAATGGGCTCGATGATAGGAGGTTTTACGTTCACATGGACCTTTAAAGTCTGAAGA P  Y  Y  P  S  Y  Y  P  P  K  C  K  C  T  W  K  F  Q  T  S       790       800       810       820       830       840CTATCAACTCTTGGCATAGCACTGAAATTCTATAACTATTCAATAACCAAGAAGAGTATGGATAGTTGAGAACCGTATCGTGACTTTAAGATATTGATAAGTTATTGGTTCTTCTCATAC L  S  T  L  G  I  A  L  K  F  Y  N  Y  S  I  T  K  K  S  M       850       860       870       880       890       900AAAGGCTGTGAGCATGGATGGTGGGAAATTTATGAGCACATGTACTGTGGCTCCTACATGTTTCCGACACTCGTACCTACCACCCTTTAAATACTCGTGTACATGACACCGAGGATGTAC K  G  C  E  H  G  W  W  E  I  Y  E  H  M  Y  C  G  S  Y  M       910       920       930       940       950       960GATCATCAGACAATTTTTCGAGTGCCCAGCCCTCTGGTTCACATTCAGCTCCAGTGCAGTCTAGTAGTCTGTTAAAAAGCTCACGGGTCGGGAGACCAAGTGTAAGTCGAGGTCACGTCA D  H  Q  T  I  F  R  V  P  S  P  L  V  H  I  Q  L  Q  C  S       970       980       990      1000      1010      1020TCAAGGCTTTCAGGCAAGCCACTTTTGGCAGAATATGGCAGTTACAACATCAGTCAACCCAGTTCCGAAAGTCCGTTCGGTGAAAACCGTCTTATACCGTCAATGTTGTAGTCAGTTGGG S  R  L  S  G  K  P  L  L  A  E  Y  G  S  Y  N  I  S  Q  P      1030      1040      1050      1060      1070      1080TGCCCTGTGGGATCTTTTAGATGCTCCTCCGGTTTATGTGTCCCTCAGGCCCAGCGTGGTACGGGACACCCTAGAAAATCTACGAGGAGGCCAAATACACAGGGAGTCCGGGTCGCACCA C  P  V  G  S  F  R  C  S  S  G  L  C  V  P  Q  A  Q  R  G      1090      1100      1110      1120      1130      1140GATGGAGTAAATGACTGCTTTGATGAAAGTGATGAACTGTTTTGCGTGAGCCCTCAACCTCTACCTCATTTACTGACGAAACTACTTTCACTACTTGACAAAACGCACTCGGGAGTTGGA D  G  V  N  D  C  F  D  E  S  D  E  L  F  C  V  S  P  Q  P      1150      1160      1170      1180      1190      1200GCCTGCAATACCAGCTCCTTCAGGCAGCATGGCCCTCTCATCTGTGATGGCTTCAGGGACCGGACGTTATGGTCGAGGAAGTCCGTCGTACCGGGAGAGTAGACACTACCGAAGTCCCTG A  C  N  T  S  S  F  R  Q  H  G  P  L  I  C  D  G  F  R  D      1210      1220      1230      1240      1250      1260TGTGAGAATGGCCGGGATGAGCAAAACTGCACTCAAAGTATTCCATGCAACAACAGAACTACACTCTTACCGGCCCTACTCGTTTTGACGTGAGTTTCATAAGGTACGTTGTTGTCTTGA C  E  N  G  R  D  E  Q  N  C  T  Q  S  I  P  C  N  N  R  T      1270      1280      1290      1300      1310      1320TTTAAGTGTGGCAATGATATTTGCTTTAGGAAACAAAATGCAAAATGTGATGGGACAGTGAAATTCACACCGTTACTATAAACGAAATCCTTTGTTTTACGTTTTACACTACCCTGTCAC F  K  C  G  N  D  I  C  F  R  K  Q  N  A  K  C  D  G  T  V      1330      1340      1350      1360      1370      1380GATTGTCCAGATGGAAGTGATGAAGAAGGCTGCACCTGCAGCAGGAGTTCCTCCGCCCTTCTAACAGGTCTACCTTCACTACTTCTTCCGACGTGGACGTCGTCCTCAAGGAGGCGGGAA D  C  P  D  G  S  D  E  E  G  C  T  C  S  R  S  S  S  A  L      1390      1400      1410      1420      1430      1440CACCGCATCATCGGAGGCACAGACACCCTGGAGGGGGGTTGGCCGTGGCAGGTCAGCCTCGTGGCGTAGTAGCCTCCGTGTCTGTGGGACCTCCCCCCAACCGGCACCGTCCAGTCGGAG H  R  I  I  G  G  T  D  T  L  E  G  G  W  P  W  Q  V  S  L      1450      1460      1470      1480      1490      1500CACTTTGTTGGATCTGCCTACTGTGGTGCCTCAGTCATCTCCAGGGAGTGGCTTCTTTCTGTGAAACAACCTAGACGGATGACACCACGGAGTCAGTAGAGGTCCCTCACCGAAGAAAGA H  F  V  G  S  A  Y  C  G  A  S  V  I  S  R  E  W  L  L  S      1510      1520      1530      1540      1550      1560GCAGCCCACTGTTTTCATGGAAACAGGCTGTCAGATCCCACACCATGGACTGCACACCTCCGTCGGGTGACAAAAGTACCTTTGTCCGACAGTCTAGGGTGTGGTACCTGACGTGTGGAG A  A  H  C  F  H  G  N  R  L  S  D  P  T  P  W  T  A  H  L      1570      1580      1590      1600      1610      1620GGGATGTATGTTCAGGGGAATGCCAAGTTTGTCTCCCCGGTGAGAAGAATTGTGGTCCACCCCTACATACAAGTCCCCTTACGGTTCAAACAGAGGGGCCACTCTTCTTAACACCAGGTG G  M  Y  V  Q  G  N  A  K  F  V  S  P  V  R  R  I  V  V  H      1630      1640      1650      1660      1670      1680GAGTACTATAACAGTCAGACTTTTGATTATGATATTGCTTTGCTACAGCTCAGTATTGCCCTCATGATATTGTCAGTCTGAAAACTAATACTATAACGAAACGATGTCGAGTCATAACGG E  Y  Y  N  S  Q  T  F  D  Y  D  I  A  L  L  Q  L  S  I  A      1690      1700      1710      1720      1730      1740TGGCCTGAGACCCTGAAACAGCTCATTCAGCCAATATGCATTCCTCCCACTGGTCAGAGAACCGGACTCTGGGACTTTGTCGAGTAAGTCGGTTATACGTAAGGAGGGTGACCAGTCTCT W  P  E  T  L  K  Q  L  I  Q  P  I  C  I  P  P  T  G  Q  R      1750      1760      1770      1780      1790      1800GTTCGCAGTGGGGAGAAGTGCTGGGTAACTGGCTGGGGGCGAAGACACGAAGCAGATAATCAAGCGTCACCCCTCTTCACGACCCATTGACCGACCCCCGCTTCTGTGCTTCGTCTATTA V  R  S  G  E  K  C  W  V  T  G  W  G  R  R  H  E  A  D  N      1810      1820      1830      1840      1850      1860AAAGGCTCCCTCGTTCTGCAGCAAGCGGAGGTAGAGCTCATTGATCAAACGCTCTGTGTTTTTCCGAGGGAGCAAGACGTCGTTCGCCTCCATCTCGAGTAACTAGTTTGCGAGACACAA K  G  S  L  V  L  Q  Q  A  E  V  E  L  I  D  Q  T  L  C  V      1870      1880      1890      1900      1910      1920TCCACCTACGGGATCATCACTTCTCGGATGCTCTGTGCAGGCATAATGTCAGGCAAGAGAAGGTGGATGCCCTAGTAGTGAAGAGCCTACGAGACACGTCCGTATTACAGTCCGTTCTCT S  T  Y  G  I  I  T  S  R  M  L  C  A  G  I  M  S  G  K  R      1930      1940      1950      1960      1970      1980GATGCCTGCAAAGGAGATTCGGGTGGACCTTTATCTTGTCGAAGAAAAAGTGATGGAAAACTACGGACGTTTCCTCTAAGCCCACCTGGAAATAGAACAGCTTCTTTTTCACTACCTTTT D  A  C  K  G  D  S  G  G  P  L  S  C  R  R  K  S  D  G  K      1990      2000      2010      2020      2030      2040TGGATTTTGACTGGCATTGTTAGCTGGGGACATGGATGTGGACGACCAAACTTTCCTGGTACCTAAAACTGACCGTAACAATCGACCCCTGTACCTACACCTGCTGGTTTGAAAGGACCA W  I  L  T  G  I  V  S  W  G  H  G  C  G  R  P  N  F  P  G      2050      2060      2070      2080      2090      2100GTTTACACAAGGGTGTCAAACTTTGTTCCCTGGATTCATAAATATGTCCCTTCTCTTTTGCAAATGTGTTCCCACAGTTTGAAACAAGGGACCTAAGTATTTATACAGGGAAGAGAAAAC V  Y  T  R  V  S  N  F  V  P  W  I  H  K  Y  V  P  S  L  L      2110      2120      2130 TAATTGCAAAAAAAAAAAAAAAAAAAAAAAATTAACGTTTTTTTTTTTTTTTTTTTTTTTProtein SequenceSequence Range 1 to 701

        10        20        30        40        50        60INLVYTTSAFSKFYEQSVVADVSSNNKGGLLVHFWIVFVMPRAKGHIFCEDCVAAILKDS        70        80        90       100       110       120IQTSIINRTSVGSLQGLAVDMDSVVLNAGLRSDYSSTIGSDKGCSQYFYAEHLSLHYPLE       130       140       150       160       170       180ISAASGRLMCHFKLVAIVGYLIRLSIKSIQIEADNCVTDSLTIYDSLLPIRSSILYRICE       190       200       210       220       230       240PTRTLMSFVSTNNLMLVTFKSPHIRRLSGIRAYFEVIPEQKCENTVLVKDITGFEGKISS       250       260       270       280       290       300PYYPSYYPPKCKCTWKFQTSLSTLGIALKFYNYSITKKSMKGCEHGWWEIYEHMYCGSYM       310       320       330       340       350       360DNQTIFRVPSPLVHIQLQCSSRLSGKPLLAEYGSYNISQPCPVGSFRCSSGLCVPQAQRG       370       380       390       400       410       420DGVNDCFDESDELFCVSPQPACNTSSFRQHGPLICDGFRDCENGRDEQNCTQSIPCNNRT       430       440       450       460       470       480FKCGNDICFRKQNAKCDGTVDCPDGSDEEGCTCSRSSSALHRIIGGTDTLEGGWPWQVSL       490       500       510       520       530       540HFVGSAYCGASVISREWLLSAAHCFHGNRLSDPTPWTAHLGMYVQGNAKFVSPVRRIVVH       550       560       570       580       590       600EYYNSQTFDYDIALLQLSIAWPETLKQLIQPICIPPTGQRVRSGEKCWVTGWGRRHEADN       610       620       630       640       650       660KGSLVLQQAEVELIDQTLCVSTYGIITSRMLCAGIMSGKRDACKGDSGGPLSCRRKSDGK       670       680       690       700WILTGIVSWGHGCGRPNFPGVYTRVSNFVPWIHKYVPSLL*

Domain organization CUB domain aa104–217 CUB domain aa222–335 LDLadomain aa340–377 LDLa domain aa381–412 LDLa domain aa415–453 Ser protdomain aa462–692

EXAMPLE 2 Expression of the Protease MTSP Domains

Nucleic acid encoding the MTSP10 and protease domain thereof can becloned into a derivative of the Pichia pastoris vector pPIC9K (availablefrom Invitrogenen; see SEQ ID NO. 13). Plasmid pPIC9k features includethe 5′ AOX1 promoter fragment at 1–948; 5′ AOX1 primer site at 855–875;alpha-factor secretion signal(s) at 949–1218; alpha-factor primer siteat 1152–1172; multiple cloning site at 1192–1241; 3′ AOX1 primer site1327–1347; 3′ AOX1 transcription termination region at 1253–1586; HIS4ORF at 4514–1980; kanamycin resistance gene at 5743–4928; 3′ AOX1fragment at 6122–6879; ColE1 origin at 7961–7288; and the ampicillinresistance gene at 8966–8106. The plasmid used herein is derived frompPIC9K by eliminating the Xhol site in the kanamycin resistance gene andthe resulting vector is herein designated pPIC9KX. Expression in Pichiacan be performed using known methods (see, e.g., Zhang et al. (2000)Biotechnology and Bioengineering 70: No 1 Oct. 5, 2000).

Mutagenesis of the Protease Domain of MTSP10 for Expression in Pichia

The gene encoding the protease domain of MTSP10 (residues 462–692 SEQ IDNo. 23) was mutagenized by PCR SOE (PCR-based splicing by overlapextension) to replace the unpaired cysteine at position 122(chymotrypsin numbering system; Cys at residue 573, SEQ ID No. 23) witha serine. Two overlapping gene fragments, each containing the AGC codonfor serine at position 122 were PCR amplified using the followingprimers: for the 5′ gene fragment,AGTTAACTCGAGAAAAGATCATCGGAGGCACAGACACCCTG SEQ ID No. 15 andACCAGTGGGAGGAATGCTTATTGGCTGAATGAG SEQ ID No. 16; for the 3′ genefragment, AGTTAAGAATTCCAAAAGAGAAGGGACATATTTATG SEQ ID No. 17 andCTCATTCAGCCAATAAGCATTCCTCCCACTGGT SEQ ID No. 18.

The amplified gene fragments were purified on a 1% agarose gel, mixedand reamplified by PCR to produce the full length coding sequence forMTSP10 C122S. This sequence was then cut with restriction enzymes EcoRIand Xhol, and ligated into vector p9KXST2. Vector p9KXST2 was derivedfrom pPic9KX with bases 1228–1233 (CCTAGG) replaced with the sequenceGGAGGTTGGTCTCATCCACAATTTGAAAAGTAA SEQ ID No. 19, which codes for ac-terminal streptagll sequence with a one glycine linker and stop codon(GGWSHPQFEK-Stop; SEQ ID No. 20).

Expression of MTSP10-ST2 C122S

P. pastoris clone GS115/pPIC9KXST2:MTSP10-ST2 C122S Sac MC5 expressingthe C122S (chymtrypsin numbering; Cys₅₇₃ in SEQ ID No. 23) mutant andC-teminally Strep-tag II tagged form of MTSP10 was fermented at the 5liter scale. An overnight culture of 200 ml (OD600 of approximately 24)was used to inoculate 3.2 liters of fermentation medium in each of sixBioflo vessels (New Brunswick Scientific, Edison, N.J.). The batch phasecomplex medium contained 10 g/l yeast extract, 20 g/l peptone, 40 g/lglycerol, 5 g/l ammonium sulfate, 0.2 g/l calcium sulfate(dihydrate), 2g/l magnesium sulfate(heptahydrate), 2 g/l potassium sulfate, 25 g/lsodium hexametaphosphate, and 4.35 ml/l PTM1. The culture was grown at apH of 5.8 and a temperature of 28° C. in the batch phase. Concentratedammonium hydroxide was used to maintain the pH of the culture. KFO 880(KABO Chemicals, Cheyenne, Wyo.) was used as needed to control foaming.

The batch phases of the fermentations lasted about 23–28 hours at whichtime the culture had consumed all of the initial glycerol in the media.A substrate limited fed-batch of 50% (w/v) glycerol was initiated at 18ml/l*hr at this point. One hour into the glycerol fed-batch the pH ofthe culture was linearly increased from 5.8 to 7.0 over a two hourperiod by addition of concentrated ammonium hydroxide. The glycerolfed-batch was from 3 to 4 hours in duration. The cultures reacheddensities of 204–234 g/l wet cell weight by this point.

Methanol induction was initiated following the end of the glycerolfed-batch phase. The culture was transitioned to methanol utilization bythe method of Zhang et al. by adding 1.5 ml of methanol per liter ofculture and linearly decreasing the glycerol feed rate from 18 ml/l*hrto 0 ml/l*hr over a 3 hour period. The methanol addition served as anon-line calibration of the MeOH Sensor (Raven Biotech, Vancouver, BC,Canada) used to control the fermenter throughout induction. After theinitial amount of methanol was utilized, as indicated by the MeOHSensor, another 1.5 ml/l was added to the culture and the MeOH sensorwas used to control the methanol concentration in the fermenter at thatlevel throughout the induction phase. The methanol fed to the fermenterwas supplemented with 2 ml/l PTM4 solution. The induction phase lastedabout 40 to 44 hours.

Recovery and Purification of MTSP-10

The supernatant from each of the fermentations was harvested afterremoval of cells by centrifugation. Supernatants were pooled andconcentrated to about 1 liter using a 10 kDa ultrafiltration cartridge(A/G Technologies Corp., Needham, Mass.) on a SRT5 ultrafiltrationsystem (North Carolina SRT, Cary, N.C.). The concentrate was diafilteredwith 3 volumes of buffer A. The concentrate was drained from the system,then the system was rinsed with a volume of buffer A equal to theconcentrated material. The concentrate and the rinse material werecombined to yield the final ultrafiltration product of about 1 liter. Afinal clarification of the supernatant was done with a SartoBran 3000.45+0.2 μm capsule filter (Sartorius Separations Div., Edgewood, N.J.).

The diafiltered MTSP10-ST2 was slowly loaded overnight onto 90 ml ofbenzamidine sepharose (Amersham Pharmacia Biotech, Piscataway, N.J.)which had been equilibrated in buffer A. The column was then washed with8 column volumes of Buffer B to remove contaminants. Elution of theMTSP10-ST2 was achieved using buffer C. Fractions of 45 ml werecollected and analyzed by activity and SDS-PAGE before pooling thedesired material to be further purified.

The MTSP10 post-benzamidine sample (usually about 150 ml containing2–5mg mtsp 10 and 100 mM benzamidine) was dialyzed into 100 mM Tris, 150mM NaCl, 1mM EDTA, 0.001% tween80 pH8.0 binding buffer. Strep-TactinMacroprep resin (Cat. No. 2-1505-010; IBA GmbH, Rudolf-Wissell-Str. 28,D-37079 Gottingen, Germany) was used to retain mtsp 10 activity. Theresin suspension was incubated in a cold room for 1 hour with shaking.The completeness of mtsp10 binding to the resin was examined bymonitoring the mtsp10 activity in the supernatant. Usually 50–70% ofmtsp 10 activity can be retained by the resin. The resin was then packedinto a column and washed with 5 column volumes of binding buffer. Mtsp10activity was eluted using binding buffer containing 2.5 mMD-desthiobiotin (Sigma product). Active fractions were identified byactivity assay using substrate Spec-tPA (Chromogenics), pooled anddialyzed to remove D-desthiobiotin.

PTM1:

6.0 g/l CuSO₄.5H₂O, 0.08 g/l NaI, 3.0 g/l MnSO₄.H₂O, 0.2 g/lNa₂MoO₄.2H₂O, 0.02 g/l H₃BO₃, 0.5 g/l CoCl₂.20.0 g/l ZnCl₂, 65.0 g/lFeSO₄.7H₂O, 0.2 g/l biotin, 5.0 ml/l H₂SO.

PTM4:

2.0 g/l CuSO₄.5H₂O, 0.08 g/l NaI, 3.0 g/l MnSO₄.H₂O, 0.2 g/lNa₂MoO₄.2H₂O, 0.02 g/l H₃BO₃, 0.5 g/l CoCl₂.6H₂O, 7.0 g/l ZnCl₂, 22.0g/l FeSO₄.7H₂O, 0.2 g/l biotin, 1.0 ml/l H₂SO₄

Buffer A: 50 mM Tris, pH 8.0, 50 mM NaCl, 0.005% Tw-80 Buffer B: 50 mMTris, pH 8.0, 1.0M NaCl, 0.005% Tw-80 Buffer C: 50 mM Tris, pH 8.0, 50mM NaCl, 100 mM benzamidine, 0.005% Tw-80

EXAMPLE 3 Assays for Identification of Candidate Compounds that Modulatethat Activity of an MTSP

Assay for Screening MTSP10 Inhibitors

The protease domain of MTSP10 expressed in Pichia pastoris was assayedfor inhibition by various compounds as follows in Costar 96 well tissueculture plates (Corning, N.Y.). Approximately 1–10 nM MTSP10 is addedwithout inhibitor, or with 100000 nM inhibitor and 7 1:6 dilutions to1×direct buffer (29.2 mM Tris, pH 8.4, 29.2 mM Imidazole, 217 mM NaCl(100 μL final volume)), and allowed to incubate at room temperature for30 minutes. 400 μM substrate Spectrozyme t-PA (American Diagnostica,Greenwich, Conn.) is added and reaction is monitored in a SpectraMAXPlus microplate reader (Molecular Devices, Sunnyvale Calif.) byfollowing change in absorbance at 405 nm for 20 minutes at 37° C.Spectrozyme UK can also be used as the substrate in this assay.

Identification of Substrates

Other substrates for use in the assays can be identified empirically bytesting substrates. The following list of substrates are exemplary ofthose that can be tested.

Substrate name Structure S 2366 pyroGlu-Pro-Arg-pNA.HCl spectrozyme t-PACH₃SO₂-D-HHT-Gly-Arg-pNA.AcOH N-p-tosyl-Gly-Pro-Arg-pNAN-p-tosyl-Gly-Pro-Arg-pNA Benzoyl-Val-Gly-Arg-pNABenzoyl-Val-Gly-Arg-pNA Pefachrome t-PA CH₃SO₂-D-HHT-Gly-Arg-pNA S 2765N-α-Z-D-Arg-Gly-Arg-pNA.2HCl S 2444 pyroGlu-Gly-Arg-pNA.HCl S 2288H-D-Ile-Pro-Arg-pNA.2HCl spectrozyme UKCbo-L-(γ)Glu(α-t-BuO)-Gly-Arg-pNA.2AcOH S 2302 H-D-Pro-Phe-Arg-pNA.2HClS 2266 H-D-Val-Leu-Arg-pNA.2HCl S 2222 Bz-Ile-Glu(g-OR)-Gly-Arg-pNA.HClR = H(50%) and R = CH₃(50%) Chromozyme PK Benzoyl-Pro-Phe-Arg-pNA S 2238H-D-Phe-Pip-Arg-pNA.2HCl S 2251 H-D-Val-Leu-Lys-pNA.2HCl Spectrozyme PIH-D-Nle-HHT-Lys-pNA.2AcOH Pyr-Arg-Thr-Lys-Arg-AMC H-Arg-Gln-Arg-Arg-AMCBoc-Gln-Gly-Arg-AMC Z-Arg-Arg-AMC Spectrozyme THEH-D-HHT-Ala-Arg-pNA.2AcOH Spectrozyme fXIIa H-D-CHT-Gly-Arg-pNA.2AcOHCVS 2081-6 (MeSO₂-dPhe-Pro-Arg-pNA) Pefachrome fVIIa(CH₃SO₂-D-CHA-But-Arg-pNA) pNA = para-nitranilide (chromogenic) AMC= amino methyl coumarin (fluorescent)

If none of the above substrates are cleaved, a coupled assay, can beused. Briefly, test the ability of the protease to activate and enzyme,such as plasminogen and trypsinogen. To perform these assays, the singlechain protease is incubated with a zymogen, such as plasminogen ortrypsinogen, in the presence of the a known substrate, such,lys-plasminogen, for the zymogen. If the single chain activates thezymogen, the activated enzyme, such as plasmin and trypsin, will degradehe substrate therefor.

EXAMPLE 4 Other Assays

These assays are described with reference to MTSP1, but such assays canbe readily adapted for use with MTSP10.

Amidolytic Assay for Determining Inhibition of Serine Protease Activityof Matriptase or MTSP1

The ability of test compounds to act as inhibitors of rMAP catalyticactivity was assessed by determining the inhibitor-induced inhibition ofamidolytic activity by the MAP, as measured by IC₅₀ values. The assaybuffer was HBSA (10 mM Hepes, 150mM sodium chloride, pH 7.4, 0.1% bovineserum albumin). All reagents were from Sigma Chemical Co. (St. Louis,Mo.), unless otherwise indicated.

Two IC₅₀ assays (a) one at either 30-minutes or 60-minutes (a 30-minuteor a 60-minute preincubation of test compound and enzyme) and (b) one at0-minutes (no preincubation of test compound and enzyme) were conducted.For the IC₅₀ assay at either 30-minutes or 60-minutes, the followingreagents were combined in appropriate wells of a Corning microtiterplate: 50 microliters of HBSA, 50 microliters of the test compound,diluted (covering a broad concentration range) in HBSA (or HBSA alonefor uninhibited velocity measurement), and 50 microliters of the rMAP(Corvas International) diluted in buffer, yielding a final enzymeconcentration of 250 pM as determined by active site filtration.Following either a 30-minute or a 60-minute incubation at ambienttemperature, the assay was initiated by the addition of 50 microlitersof the substrate S-2765(N-α-Benzyloxycarbonyl-D-arginyl-L-glycyl-L-arginine-p-nitroanilinedihydrochloride; DiaPharma Group, Inc.; Franklin, Ohio) to each well,yielding a final assay volume of 200 microliters and a final substrateconcentration of 100 μM (about 4-times K_(m)). Before addition to theassay mixture, S-2765 was reconstituted in deionized water and dilutedin HBSA. For the IC₅₀ assay at 0 minutes; the same reagents werecombined: 50 microliters of HBSA, 50 microliters of the test compound,diluted (covering the identical concentration range) in HBSA (or HBSAalone for uninhibited velocity measurement), and 50 microliters of thesubstrate S-2765. The assay was initiated by the addition of 50microliters of rMAP. The final concentrations of all components wereidentical in both IC₅₀ assays (at 30- or 60- and 0-minute).

The initial velocity of chromogenic substrate hydrolysis was measured inboth assays by the change of absorbance at 405 nM using a Thermo Max®Kinetic Microplate Reader (Molecular Devices) over a 5 minute period, inwhich less than 5% of the added substrate was used. The concentration ofadded inhibitor, which caused a 50% decrease in the initial rate ofhydrolysis was defined as the respective IC₅₀ value in each of the twoassays (30- or 60-minutes and 0-minute).

In Vitro Enzyme Assays for Specificity Determination

The ability of compounds to act as a selective inhibitor of matriptaseactivity was assessed by determining the concentration of test compoundthat inhibits the activity of matriptase by 50%, (IC₅₀) as described inthe above Example, and comparing IC₅₀ value for matriptase to thatdetermined for all or some of the following serine proteases: thrombin,recombinant tissue plasminogen activator (rt-PA), plasmin, activatedprotein C, chymotrypsin, factor Xa and trypsin.

The buffer used for all assays was HBSA (10 mM HEPES, pH 7.5, 150 mMsodium chloride, 0.1% bovine serum albumin).

The assay for IC₅₀ determinations was conducted by combining inappropriate wells of a Corning microtiter plate, 50 microliters of HBSA,50 microliters of the test compound at a specified concentration(covering a broad concentration range) diluted in HBSA (or HBSA alonefor V₀ (uninhibited velocity) measurement), and 50 microliters of theenzyme diluted in HBSA. Following a 30 minute incubation at ambienttemperature, 50 microliters of the substrate at the concentrationsspecified below were added to the wells, yielding a final total volumeof 200 microliters. The initial velocity of chromogenic substratehydrolysis was measured by the change in absorbance at 405 nm using aThermo Max® Kinetic Microplate Reader over a 5 minute period in whichless than 5% of the added substrate was used. The concentration of addedinhibitor which caused a 50% decrease in the initial rate of hydrolysiswas defined as the IC₅₀ value.

Thrombin (fIIa) Assay

Enzyme activity was determined using the chromogenic substrate,Pefachrome t-PA(CH₃SO₂-D-hexahydrotyrosine-glycyl-L-Arginine-p-nitroaniline, obtainedfrom Pentapharm Ltd.). The substrate was reconstituted in deionizedwater prior to use. Purified human α-thrombin was obtained from EnzymeResearch Laboratories, Inc. The buffer used for all assays was HBSA (10mM HEPES, pH 7.5, 150 mM sodium chloride, 0.1% bovine serum albumin).

IC₅₀ determinations were conducted where HBSA (50 μL), α-thrombin (50μl) (the final enzyme concentration is 0.5 nM) and inhibitor (50 μl)(covering a broad concentration range), were combined in appropriatewells and incubated for 30 minutes at room temperature prior to theaddition of substrate Pefachrome-t-PA (50 μl) (the final substrateconcentration is 250 μM, about 5 times Km). The initial velocity ofPefachrome t-PA hydrolysis was measured by the change in absorbance at405 nm using a Thermo Max® Kinetic Microplate Reader over a 5 minuteperiod in which less than 5% of the added substrate was used. Theconcentration of added inhibitor which caused a 50% decrease in theinitial rate of hydrolysis was defined as the IC₅₀ value.

Factor Xa

Factor Xa catalytic activity was determined using the chromogenicsubstrate S-2765(N-benzyloxycarbonyl-D-arginine-L-glycine-L-arginine-p-nitroaniline),obtained from DiaPharma Group (Franklin, Ohio). All substrates werereconstituted in deionized water prior to use. The final concentrationof S-2765 was 250 μM (about 5-times Km). Purified human Factor X wasobtained from Enzyme Research Laboratories, Inc. (South Bend, Ind.) andFactor Xa (FXa) was activated and prepared from it as described [Bock,P. E., Craig, P. A., Olson, S. T., and Singh, P. Arch. Biochem. Biophys.273:375–388 (1989)]. The enzyme was diluted into HBSA prior to assay inwhich the final concentration was 0.25 nM. Recombinant tissueplasminogen activator (rt-PA) Assay

rt-PA catalytic activity was determined using the substrate, Pefachromet-PA (CH₃SO₂-D-hexahydrotyrosine-glycyl-L-arginine-p-nitro-aniline,obtained from Pentapharm Ltd.). The substrate was made up in deionizedwater followed by dilution in HBSA prior to the assay in which the finalconcentration was 500 micromolar (about 3-times Km). Human rt-PA(Activase®) was obtained from Genentech Inc. The enzyme wasreconstituted in deionized water and diluted into HBSA prior to theassay in which the final concentration was 1.0 nM.

Plasmin Assay

Plasmin catalytic activity was determined using the chromogenicsubstrate, S-2366 (L-pyroglutamyl-L-prolyl-L-arginine-p-nitroanilinehydrochloride), which was obtained from DiaPharma group. The substratewas made up in deionized water followed by dilution in HBSA prior to theassay in which the final concentration was 300 micromolar (about2.5-times Km). Purified human plasmin was obtained from Enzyme ResearchLaboratories, Inc. The enzyme was diluted into HBSA prior to assay inwhich the final concentration was 1.0 nM.

Activated Protein C (aPC) Assay

aPC catalytic activity was determined using the chromogenic substrate,Pefachrome PC(delta-carbobenzloxy-D-lysine-L-prolyl-L-arginine-p-nitroanilinedihydrochloride), obtained from Pentapharm Ltd.). The substrate was madeup in deionized water followed by dilution in HBSA prior to the assay inwhich the final concentration was 400 micromolar (about 3-times Km).Purified human aPC was obtained from Hematologic Technologies, Inc. Theenzyme was diluted into HBSA prior to assay in which the finalconcentration was 1.0 nM.

Chymotrypsin Assay

Chymotrypsin catalytic activity was determined using the chromogenicsubstrate, S-2586(methoxy-succinyl-L-arginine-L-prolyl-L-tyrosyl-p-nitroanilide), whichwas obtained from DiaPharma Group. The substrate was made up indeionized water followed by dilution in HBSA prior to the assay in whichthe final concentration was 100 micromolar (about 9-times Km). Purified(3×-crystallized; CDI) bovine pancreatic alpha-chymotrypsin was obtainedfrom Worthington Biochemical Corp. The enzyme was reconstituted indeionized water and diluted into HBSA prior to assay in which the finalconcentration was 0.5 nM.

Trypsin Assay

Trypsin catalytic activity was determined using the chromogenicsubstrate, S-2222 (benzoyl-L-isoleucine-L-glutamic acid-[gamma-methylester]-L-arginine-p-nitroanilide), which was obtained from DiaPharmaGroup. The substrate was made up in deionized water followed by dilutionin HBSA prior to the assay in which the final concentration was 250micromolar (about 4-times Km). Purified (3×-crystallized; TRL3) bovinepancreatic trypsin was obtained from Worthington Biochemical Corp. Theenzyme was reconstituted in deionized water and diluted into HBSA priorto assay in which the final concentration was 0.5 nM.

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

1. A substantially purified single or two chain protease comprising anamino acid sequence having at least 99% identify to the sequence of theMTSP10protease amino acid sequence set forth in SEQ ID No.
 23. 2. Thesubstantially purified MTSP10 protease of claim 1 which is a two chainprotease.
 3. The substantially purified MTSP10 protease of claim 1 whichis a single chain protease.
 4. The MTSP10 protease of claim 1 wherein afree cysteine in the protease domain is replaced with another aminoacid.
 5. The MTSP10 protease of claim 4, wherein the replacing aminoacid is a serine.
 6. The substantially purified protease of claim 1,wherein catalytic activity includes in vitro proteolysis ofCH₃SO₂-D-HHT-Gly-Arg-pNA.AcOH (spectrozyme t-PA).
 7. A substantiallypurified single or two chain MTSP10 protease selected from the groupconsisting of a protease that comprises the sequence of amino acidresidues set forth in SEQ ID No. 23 and a MTSP10 protease that comprisesa sequence of amino acid residues encoded by a nucleic acid moleculehaving the sequence of nucleotides set forth in SEQ ID No.
 22. 8. Thesubstantially purified MTSP10 protease of claim 7 which is a two chainprotease.
 9. The substantially purified protease of claim 7, whereincatalytic activity includes in vitro proteolysis ofCH₃SO₂-D-HHT-Gly-Arg-pNA.AcOH (spectrozyme t-PA).
 10. The protease ofclaim 7 that consists of the sequence of amino acid residues set forthin SEQ ID No.
 23. 11. A substantially purified single or two chainMTSP10 protease that comprises one or more amino acid substitutions atpositions selected from the group of positions consisting of 67, 272,336, 383, 409, 418, 462–467 and 573 in the sequence set forth in SEQ IDNo.
 23. 12. A substantially purified MTSP10 protease that consists ofthe sequence of amino acids at positions 1–230 of SEQ ID No.
 6. 13. Asubstantially purified single chain MTSP10 protease that comprises anamino acid substitution at one or more positions selected from the groupof positions consisting of 1–6 or 112 of SEQ ID No.
 6. 14. Asubstantially purified two chain MTSP10 protease that comprises an aminoacid substitution at one or more positions selected from the group ofpositions consisting of 1–6 or 112 of SEQ ID No.
 6. 15. A conjugate,comprising the protease of claim 1, and a targeting agent linked to theprotease directly or via a linker.
 16. The conjugate of claim 15,wherein the targeting agent permits affinity isolation or purificationof the conjugate; attachment of the conjugate to a surface; detection ofthe conjugate; or targeted delivery of the conjugate to a selectedtissue or cell.
 17. A conjugate comprising the protease of claim 11 anda targeting agent linked to the protease directly or via a linker. 18.The conjugate of claim 17, wherein the targeting agent permits affinityisolation or purification of the conjugate, attachment of the conjugateto a surface; detection of the conjugate; or targeted delivery of theconjugate to a selected tissue or cell.
 19. A conjugate comprising theprotease of claim 12 and a targeting agent linked to the proteasedirectly or via a linker.
 20. The conjugate of claim 19, wherein thetargeting agent permits affinity isolation or purification of theconjugate; attachment of the conjugate to a surface; detection of theconjugate; or targeted delivery of the conjugate to a selected tissue orcell.
 21. A solid support comprising two or more proteases of claim 1linked thereto either directly or via a linker.
 22. The support of claim21, wherein the proteases comprise an array.
 23. The support of claim22, wherein the array further comprises a plurality of differentprotease domains.
 24. A method for identifying compounds that inhibitthe protease activity of a MTSP10 protease of claim 1 or claim 11comprising: contacting a protease of claim 1 or claim 11 with asubstrate that is proteolytically cleaved by the protease, and, eithersimultaneously, before or after, adding a test compound or pluralitythereof; measuring the amount of substrate cleaved in the presence ofthe test compound; and selecting test compounds that decrease the amountof substrate cleaved compared to a control, thereby identifyingcompounds that inhibit the activity of the protease.
 25. The method ofclaim 24, wherein the test compounds are small molecules, peptides,peptidomimetics, natural products, antibodies or fragments thereof thatinhibit the activity of the protease.
 26. The method of claim 24,wherein a plurality of the test compounds are screened simultaneously.27. The method of claim 24, wherein the control measures the amount ofsubstrate cleaved in the absence of the test compound.
 28. The method ofclaim 26, wherein a plurality of the proteases are linked to a solidsupport, either directly or via a linker.
 29. The method of claim 28,wherein the proteases comprise an array.
 30. A method for identifyingcompounds that inhibit the protease activity of the MTSP10 protease ofclaim 2, comprising: contacting the MTSP10 protease of claim 2 with asubstrate that is proteolytically cleaved by the protease, and, eithersimultaneously, before or after, adding a test compound or pluralitythereof; measuring the amount of substrate cleaved in the presence ofthe test compound; and selecting test compounds that decrease the amountof substrate cleaved compared to a control, thereby identifyingcompounds that inhibit the activity of the protease.
 31. A method foridentifying test compounds that inhibit the protease activity of theMTSP10 protease of claim 7 comprising: contacting the MTSP10 protease ofclaim 7 with a substrate proteolytically cleaved by the protease, and,either simultaneously, before or after, adding a test compound orplurality thereof; measuring the amount of substrate cleaved in thepresence of the test compound; and selecting test compounds thatdecrease the amount of substrate cleaved compared to a control, therebyidentifying compounds that inhibit the activity of the protease.
 32. Amethod for identifying test compounds that inhibit the protease activityof a the MTSP10 protease of claim 8, comprising: contacting the MTSP10protease of claim 8 with a substrate that is proteolytically cleaved bythe protease; and, either simultaneously, before or after, adding a testcompound or plurality thereof; measuring the amount of substrate cleavedin the presence of the test compound; and selecting test compounds thatdecrease the amount of substrate cleaved compared to a control, therebyidentifying compounds that inhibit the activity of the protease.
 33. Amethod for identifying test compounds that inhibit the protease activityof the MTSP10 protease of claim 12, comprising: contacting a the MTSP10protease of claim 12 with a substrate that is proteolytically cleaved bythe protease; and, either simultaneously, before or after, adding a testcompound or plurality thereof; measuring the amount of substrate cleavedin the presence of the test compound; and selecting test compounds thatdecrease the amount of substrate cleaved compared to a control, therebyidentifying compounds that inhibit the activity of the protease.
 34. Amethod of identifying a compound that specifically binds to asingle-chain and/or two-chain MTSP10 protease of claim 1 or claim 11,comprising: contacting an MTSP10 protease of claim 1 or claim 11 with atest compound or plurality thereof under conditions conducive to bindingof the test compound to the protease; and selecting test compounds thatremain bound specifically to the protease compared to a control, therebyidentifying test compounds that specifically bind a single chain and/ortwo chain form of the protease.
 35. The method of claim 34, wherein theprotease is linked either directly or indirectly via a linker to a solidsupport.
 36. The method of claim 34, wherein the test compounds aresmall molecules, peptides, peptidomimetics, natural products, antibodiesor fragments thereof.
 37. The method of claim 34, wherein a plurality ofthe test compounds are screened simultaneously.
 38. The method of claim34, wherein a plurality of the proteases are linked to a solid support.39. A method of identifying a compound that binds to the single-chainand/or two-chain form of the MTSP10 protease of claim 7, comprising:contacting the MTSP10 protease of claim 7 with a test compound orplurality thereof under conditions conducive to binding of the testcompound to the protease; and selecting test compounds that remain boundspecifically to the protease compared to a control, thereby identifyingtest compounds that specifically bind a single chain and/or two chainform of the protease.
 40. A method for identifying activators of thezymogen form of an MTSP10 protease of claim 1 or claim 11, comprising:contacting a zymogen form of an MTSP10 protease of claim 1 or claim 11with a substrate of the activated form of the protease; detectingcleavage of the substrate, thereby identifying compounds that activatethe zymogen.
 41. The method of claim 40, wherein the substrate is achromogenic substrate.
 42. The method of claim 40, wherein the substrateis a L-pyroglutamyl-L-prolyl-L-arginine-p-nitroaniline hydrochloride.43. The method of claim 40, wherein the test compound is a smallmolecule, a nucleic acid or a protease.